Methods of identifying agents which bind GPR-9-6

ABSTRACT

The invention relates to an antibody or antigen-binding fragment thereof which binds to the CC chemokine receptor GPR-9-6 and blocks the binding of a ligand (e.g., TECK) to the receptor. The invention also relates to a method of identifying agents (molecules, compounds) which can bind to GPR-9-6 and inhibit the binding of a ligand (e.g., TECK) and/or modulate a function of GPR-9-6. The invention further relates to a method of modulating a function of GPR-9-6, and to the use of the antibodies, antigen-binding fragments and agents identified by the method of the invention in research, therapeutic, prophylactic and diagnostic methods.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/266,464,filed Mar. 11, 1999, now U.S. Pat. No. 6,329,159 B1. The entireteachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Chemokines are a large and growing family of nearly forty 6-14 kD(non-glycosylated) heparin binding proteins that mediate a wide range ofbiological functions (Taub, D. D. and Openheim, J. J., Ther. Immunol.,1:229-246 (1994)). The chemokines can be divided into families based onthe position of four cysteine residues that form two disulfide bonds(Kelner, G. S., et al., Science, 266:12395-1399 (1994); Bazan, J. F., etal., Nature, 385:640-644 (1997); Pin, Y., et al., Nature, 385:611-617(1997)). Chemokine receptors can also be divided into families based onthe type of chemokine they bind, although, no clear structuraldifferences have been identified that distinguish the receptorsub-families (Mackay, C. R., J. Exp. Med., 184:799-802 (1996)). Inaddition, there are a number of so called “orphan” chemokine receptors(e.g., GPR-9-6) which share sequence homology with well characterizedchemokine receptors. However, the biological functions and specificagonists of orphan receptors remain unknown.

Chemokines play a vital role in leukocyte adhesion and extravasation.For example, in various in vitro assays, chemokines can induce thechemotaxis or transendothelial migration of leukocytes (Taub, D. D. andOpenheim, J. J., Ther. Immunol., 1:229-246 (1994)), while in vivoinjection (Taub, D. D., et al., J. Clin. Invest., 97:1931-1941 (1996))or over-expression of chemokines (Fuentes, M. E., et al., J. Immunol.,155:5769-5776 (1995)) can result in leukocyte accumulation at the siteof chemokine injection or expression, Antagonists of chemokines canprevent leukocyte trafficking (Bargatze, R. F. and Butcher, E. C., J.Exp. Med., 178:367-372 (1993)) and may have beneficial effects inseveral models of acute and chronic inflammation (Sekido, N., et al.,Nature, 365:654-657 (1993); Karpus, W. J., et al., J. Immunol.,155:5003-5010 (1995)). Chemokines have also been reported to modulateangiogenesis (Gupta, S. K., et at, Proc. Natl. Acad. Sci. USA,92:7799-7803 (1995)), hematopoiesis (Taub, D. D. and Openheim, J. J.,Ther. Immunol., 1:229-246 (1994)) as well as T lymphocyte activation(Zhou, Z., et al., J. Immunol. 151:4333-4341 (1993); Taub, D. D., etal., J. Immunol., 156:2095-2103 (1996)). In addition, several chemokinereceptors act as co-receptors, along with CD4, for entry of M tropic andT tropic HIV-1 (Choe, H., et al., Cell, 85:1135-1148 (1996); Feng, Y.,et al., Science, 272:872-877 (1996)).

Several subsets of CD4 lymphocytes can be defined based on theirexpression of various adhesion molecules that are known to effecttrafficking to different physiologic sites (Mackay, C. R., Curr. Opin.Immunol., 5:423-427 (1993)). For example, CLA^(+ve) memory CD4lymphocytes traffic to the skin (Berg, E. L., et al., Nature, 174(6):1461-1466 (1991)), while CLA^(−ve) α4β7^(+ve) memory CD4 lymphocytestraffic to mucosal sites (Hamman, A., et al., J. Immunol., 152:3282-3292(1994)). Leukocyte adhesion to endothelium is thought to involve severaloverlapping steps including rolling, activation and arrest. Rollingleukocytes are exposed to factors expressed at the adhesion siteresulting in activation of the leukocyte and up-regulation ofintegrin-mediated adhesion. As a consequence of such integrin-mediatedinteractions, leukocytes arrest on the endothelium (Bargatze, R. F. andButcher, L. C., J. Exp. Med., 178:367-372 (1993); Bargatze, R. F., etal., Immunity, 3:99-108 (1995)). Leukocyte activation and up-regulationof integrin molecules occurs via a peltussis toxin sensitive mechanismthat is thought to involve chemokine receptors (Bargatze, R. F. andButcher, E. C., J. Exp. Med., 178:367-372 (1993); Campbell, J. J., etal., Science, 279:381-383 (1998)).

Memory CD4⁺ lymphocytes can be grouped based upon the expression ofcertain chemokine receptors. For example, CXCR3, CCR2 and CCR5 (Qin, S.,et al., Eur. A Immunol., 26:640-647 (1996); Qin, S., et al., J. Clin.Invest., 101:746-754 (1998); Liao, F., et al., J. Immunol., 162:186-194(1999)) are all expressed on subsets of memory CD4 lymphocytes, andcertain chemokines act selectively on naive T cells (Adema, G. J., etal., Nature, 387:713-717 (1997)). Furthermore, several chemokines whichare ligands for such receptors have been shown to be expressed ininflammatory sites (Gonzalo, J. A., et al., J. Clin. Invest.,98:2332-2345 (1996)) and in some cases in lymph nodes draining achallenged site (Tedla, N., et al., J. Immunol., 161:5663-5672 (1998)).In vitro derived T_(H)1/T_(H)2 lymphocyte lines have also been shown todifferentially express chemokine receptors. Specifically, T_(H)1lymphocytes have been shown to selectively express CXCR3 and CCR5, whileT_(H)2 lymphocytes selectively express CCR4, CCR8 and CCR3 (Bonecchi, R.G., et al., J. Exp. Med., 187:129-134 (1998); Sallusto, F. D., et al.,J. Exp. Med., 187:875-883 (1998); Sallusto, F., Science, 277:2005-2007(1997); Andrew, D. P., et al., J. Immunol 161:5027-5038 (1998); Zingoni,A., et al., J. Immunol., 161:547-555 (1998)). Interestingly, in somecases the chemokines for these respective chemokine receptors, such asMDC for CCR4 and IP-10 for CXCR3, are induced by cytokines associatedwith a T_(H)1/T_(H)2 environment (Andrew, D. P., et al., J. Immunol161:5027-5038(1998); Luster, A. D., et al., Nature, 315:672-676 (1985)).

SUMMARY OF THE INVENTION

The invention relates to an antibody (immunoglobulin) or functionalfragment thereof (e.g., an antigen-binding fragment) which binds to amammalian GPR-9-6 or portion of the receptor. In one embodiment, theantibody or antigen-binding fragment thereof binds to human GPR-9-6. Inanother embodiment, the antibody or antigen-binding fragment thereof caninhibit the binding of a ligand to a mammalian GPR-9-6. In a preferredembodiment, the antibody or antibody-binding fragment can bind to humanGPR-9-6 and inhibit the binding of TECK to the receptor.

In another embodiment, the antibody or antigen-binding fragment of theinvention binds to an epitope which is the same as or is similar to theepitope recognized by mAb 3C3 or an antigen-binding fragment thereof.For example, the binding of the antibody or antigen-binding fragment ofthe invention to human GPR-9-6 can be inhibited a peptide that consistsof the amino acid sequence of SEQ ID NO:3. In another embodiment, thebinding of the antibody or antigen-binding fragment of the invention tohuman GPR-9-6 can be inhibited by mAb 3C3. In a preferred embodiment,the antibody is mAb 3C3 or antigen-binding fragment thereof.

The invention also relates to an isolated cell that produces an antibodyor antigen-binding fragment of the present invention, including thosewhich bind to mammalian GPR-9-6 and inhibit the binding of a ligand tothe receptor. In a particular embodiment, the isolated cell is murinehybridoma 3C3 (also referred to as murine hybridoma LS129-3C3-E3-1)deposited under ATCC Accession No. HB-12653.

The invention also relates to a method of detecting or identifying anagent (i.e., molecule or compound) which binds to a mammalian GPR-9-6.In one embodiment, an agent which can bind to mammalian GPR-9-6 andinhibit (reduce or prevent) the binding of a ligand (e.g., TECK) toGPR-9-6 is identified in a competitive binding assay. In otherembodiments, agents for use in therapy are identified in a directbinding assay. Thus, the invention encompasses methods of identifyingagents which modulate GPR-9-6 function, such as, ligands or othersubstances which bind a mammalian GPR-9-6, including inhibitors (e.g.,antagonists) or promoters (e.g., agonists) of receptor function. Asuitable source of a mammalian GPR-9-6 or a ligand-binding variantthereof can be used to identify a GPR-9-6 binding agent in accordancewith the method of the invention. In one embodiment, a cell (e.g., cellline, recombinant cell) that expresses a mammalian GPR-9-6 or a ligandbinding variant thereof is used. In another embodiment, a membranepreparation of a cell that expresses a mammalian GPR-9-6 or a ligandbinding variant thereof is used.

The invention also relates to therapeutic methods in which agents whichcan bind to a mammalian GPR-9-6 and modulate (inhibit or promote) aGPR-9-6 function are administered to a subject in need of such therapy.In one embodiment, the therapeutic method is a method of treating asubject having an inflammatory disease. In a preferred embodiment, thesubject has an inflammatory diseases associated with mucosal tissues,such as an inflammatory bowel disease. In a particular embodiment, theinflammatory bowel disease is Crohn's disease or colitis. In anotherembodiment, the therapeutic method is a method of inhibitingGPR-9-6-mediated homing of leukocytes. In another embodiment, the methodis a method of modulating a GPR-9-6 function.

The invention further relates to a method for detecting or quantifying amammalian GPR-9-6 or a portion thereof in a biological sample. Themethod comprises contacting a biological sample and an anti-GPR-9-6antibody or antigen-binding fragment of the invention under conditionssuitable for binding, and detecting a complex formed between GPR-9-6 andthe antibody or antigen-binding fragment. In one embodiment thebiological sample comprises human cells or a fraction of said cells(e.g., membrane preparation).

The invention also relates to a test kit for identifying or quantifyinga mammalian GPR-9-6 or a portion thereof in a biological sample. In oneembodiment, the kit comprises an antibody of the invention and suitableancillary reagents.

The present invention farther relates to an antibody, antigen-bindingfragment or agent as described herein for use in therapy (includingprophylaxis) or diagnosis, and to the use of such an antibody,antigen-binding fragment or agent for the manufacture of a medicamentfor the treatment of a particular disease or condition as describedherein (e.g., an inflammatory disease associated with mucosal tissues).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a dendrogram illustrating the relationship of GPR-9-6 to otherleukocyte chemokine receptors. Using a clustal alignment analysisprogram (DNAstar), the protein sequences of leukocyte chemokinereceptors were aligned and used to determine the phylogenetic distancesbetween GPR-9-6 and several chemokine receptors.

FIGS. 2A-2B illustrate the specific binding of mAb 3C3 to GPR-9-6transfectants. In FIG. 2A, GPR-9-6/L1.2 transfectants were stained withmAb 3C3 (stippled profile), anti-CCR6 antibody ( ^(. . .) ) or with amurine IgG2b mAb ( - - - ) (n=2). In FIG. 2B, CCR6/L1.2 transfectantswere stained with mAb 3C3 ( ^(. . .) ), anti-CCR6 antibody (stippledprofile) or with a murine IgG2b mAb ( - - - ) (n=2).

FIGS. 3A-3I are a series of fluorescence plots which illustrate thatGPR-9-6 is expressed on B lymphocytes and subsets of CD4 and CD8lymphocytes. mAb 3C3 was used in two color studies on mononuclear cellsalong with anti-CD4 FITC (FIG. 3A), anti-CD8 FITC (FIG. 3B), anti-CD19FITC (FIG. 3C), anti-CD56 Cychrome (FIG. 3D) and anti-CCR3 FITC (FIG.3E). For thymocytes (FIG. 3F), two color studies were performed with mAb3C3 and anti-TcR Cychrome. GPR-9-6 expression on monocytes (FIG. 3G),eosinophils (FIG. 3H) and neutrophils (FIG. 3I) was evaluated in onecolor studies using isolated populations of these cells and mAb 3C3 (-)and IgG2b controls ( - - - ). Anti-CCR2, anti-CCR3 and anti-CXCR2antibodies were used as positive controls for monocytes, eosinophils andneutrophils, respectively (stippled profiles) (n=3).

FIGS. 4A-4H are plots illustrating that GPR-9-6 is not expressed onimmature dendritic cells (IMDC), mature dendritic cells (MDC) orT_(H)1/T_(H)2 lymphocytes. Mature (-) and immature dendritic cells(stippled profile) were stained with anti-CCR5 (FIG. 4A), anti-CD83(FIG. 4B), anti-CD86 (FIG. 4C) or anti-GPR-9-6 (FIG. 4D). Staining withIgG2b control on IMDCs ( ^(. . .) ) is also shown. FIG. 4E showsstaining of umbilical CD4 lymphocytes with anti-CXCR4 (stippledprofile), anti-GPR-9-6 (-) and IgG2b ( ^(. . .) ). FIGS. 4F-4H showstaining of T_(H)1 (stippled profiles) and T_(H)2 (-) lymphocytes withanti-CXCR3 (FIG. 4F), anti-α4β7 (Act1) (FIG. 4G) or anti-GPR-9-6 (mAb3C3) (FIG. 4H) as indicated, with ( ^(. . .) ) representing stainingwith an IgG2b control on T_(H)1 lymphocytes (n=3).

FIGS. 5A-5C are graphs illustrating the modulation of GPR-9-6 onlymphocytes over time and upon T lymphocyte activation. Mononuclearcells were isolated from one individual at set times over 14 days andstained in two color experiments using mAb 3C3 and anti-CD4 FITC oranti-CD19 FITC to examine GPR-9-6 expression on B and CD4 lymphocytes(FIG. 5A). In FIGS. 5B-5C, mononuclear cells were activated with platebound anti-TcR mAb OKT3 for 4 days, followed by expansion with IL-2 at 5ng/ml. Aliquots of cells were stained over time with mAb 3C3 (FIG. 5B)to determine GPR-9-6 expression upon T lymphocyte activation, or withanti-CCR6 mAb and anti-CCR5 mAb (FIG. 5C) to determine expression ofCCR-3 and CCR-5 upon T cell activation. (n=2)

FIGS. 6A-6F are a series of fluorescence plots illustrating that GPR-9-6is expressed on α4β7^(high) CLA^(−ve) CD4⁺ memory lymphocytes.Mononuclear cells were stained in three color experiments using anti-CD4cychrome to gate on CD4 lymphocytes. The cells were also stained withanti-GPR-9-6 mAb 3C3 followed by F(ab′)₂ anti-mouse IgG phycoerythrin tostudy GPR-9-6 expression on subsets defined with anti-αE (HML1, BeckmanCoulter, Inc., Fullerton, Calif.) (FIG. 6A), anti-β7 (Fib504,PharMingen, San Diego, Calif.) (FIG. 6B), anti-CD49d (HP2/1, PharMingen,San Diego, Calif.) (FIG. 6C), anti-CLA (HECA 452, PharMingen, San Diego,Calif.) (FIG. 6D), anti-CD45RO (UCLH1, PharMingen, San Diego, Calif.)(FIG. 6E) and anti-CD62L (CD56)(PharMingen, San Diego, Calif.) (FIG. 6F)(n=5).

FIGS. 7A-7F are a series of fluorescence plots illustrating theexpression of GPR-9-6 on CD4 lymphocytes in relation to other chemokinereceptors. Mononuclear cells were stained in three-color experimentsusing anti-CD4 cychrome to gate on CD4 lymphocytes. The cells were alsostained with anti-GPR-9-6 mAb 3C3 followed by F(ab′)₂ anti-mouse IgGcoupled to phycoerythrin to study GPR-9-6 expression on subsets definedwith anti-CCR2 (R&D Systems, Minneapolis, Minn.) (FIG. 7A), anti-CCR5(PharMingen, San Diego, Calif.) (FIG. 7B), anti-CCR6 (R&D Systems,Minneapolis, Minn.) (FIG. 7C), anti-CXCR3 (1C6, Leukosite, Inc.,Cambridge, Mass.) (FIG. 7D), anti-CXCR4 (PharMingen, San Diego, Calif.)(FIG. 7E) and anti-CXCR5 (R&D Systems, Minneapolis, Minn.) (FIG. 7F),all of which were coupled to phycoerythrin (n=2).

FIGS. 8A-8F are a graph and series of histograms illustrating thatGPR-9-6 is a chemokine receptor for TECK. GPR-9-6/L1.2 transfectantswere tested for a chemotactic response to 10 to 1000 nM TECK (FIG. 8A).FIG. 8B shows that anti-GPR-9-6 (mAb 3C3) inhibited 150 nM TECK-inducedchemotaxis of GPR-9-6/L1.2 transfectants, while anti-CCR3 does not. FIG.8C illustrates that pertussis toxin (PTX) pretreatment of theGPR-9-6/L1.2 transfectants inhibited 150 nM TECK-induced chemotaxis ofGPR-9-6/L1.2 transfectants. FIG. 8D and FIG. 8E illustrate the abilityof MOLT-4 cells and the inability of SKW3 cells, respectively, tochemotax to TECK. FIG. 8F illustrates the ability of MOLT-13 cells tochemotax in response to 150 nM TECK, and the ability of mAb 3C3 to blockthis migration, using SDF1α at 100 ng/ml as a chemokine known to inducechemotaxis of these cells through CXCR4 (n=2).

FIGS. 9A-9C illustrate that GPR-9-6 expressing cell lines undergo Ca²⁺flux in response to TECK. The GPR-9-6 expressing cell line MOLT-4 wasloaded with the Ca²⁺ sensitive dye Fura-2 and then tested for theirability to mobilize Ca²⁺ in response to 150 nM TECK (FIG. 9A), 100 nMSDF1α (FIG. 9B) or 100 nM MDC (FIG. 9C) chemokines (n=2).

FIGS. 10A-10F are a series of histograms illustrating that a subset ofCD4 lymphocytes and thymocytes chemotax to TECK. CD4⁺ lymphocytes (FIG.10F), CD8⁺ lymphocytes (FIG. 10B), CD56⁺ NK cells (FIG. 10D) and CD14⁺monocytes (FIG. 10A) were isolated from mononuclear cells using theappropriate Miltenyi Beads. Neutrophils (FIG. 10E) were isolated bydextran precipitation followed by Ficoll and eosinophils (FIG. 10C)separated from neutrophils by depletion with anti-CD16 Miltenyi Beads.Uncoated 3 μm Costar plates were used to assess chemotaxis with theseleukocyte subsets, with the exception of eosinophils and neutrophils,for which ECV304 monolayers were grown over the inserts before theassay. In each case, TECK was tested in a dose response fashion between1 nM and 220 nM. Chemokines known to act on the leukocyte subsets wereused as positive controls (n=2).

FIGS. 11A-11C are a series of histograms illustrating that TECK-inducedchemotaxis of thymocytes and CD4 lymphocytes is mediated by GPR-9-6. CD4lymphocytes and thymocytes were pre-treated with anti-GPR-9-6 mAb 3C3 at50 μg/ml before use in chemotaxis assays. Thymocytes were assayed using150 nM TECK and 100 nM SDF1α (FIG. 11A), CD4 lymphocytes were assayedusing 150 nM TECK (FIG. 11B), and CD4 lymphocytes were assayed using 100nM TARC (FIG. 11C). In all assays, TECK-induced chemotaxis was inhibitedby anti-GPR-9-6 (mAb 3C3). Irrelevant mAb anti-CCR6 mAb 2A9 (FIG. 11A)and anti-CCR4 mAb 2B10 (FIG. 11B) were also examined for their effect onCD4 or thymocyte chemotaxis to TECK or TARC. For CD4 lymphocytechemotaxis the effect of mAb 3C3 on TARC induced CD4 lymphocytechemotaxis was also tested (FIG. 11C) as a further negative control(n=2).

FIGS. 12A-12C illustrate the tissue distribution of TECK and GPR-9-6.Multi-tissue Northern blot analysis filters (2 μg RNA/lane) (ClonTech)and a Northern blot prepared using RNA from various cell lines (20μg/lane) were probed with ³²P TECK DNA probes (FIG. 12A) or labeledGPR-9-6 (FIG. 12B) to determine their tissue distribution. In FIG. 12C,cDNA (ClonTech) from colon, small intestine, brain, lymph node, spleen,thymus, and genomic DNA were amplified in PCR (30 cycles) using primersdesigned from the sequence of GPR-9-6.

FIGS. 13A-13B are histograms illustrating that only α4β7^(high) CD4 andCD8 lymphocytes migrate to TECK. In a 4 color sort, memory CD8lymphocytes defined by intermediate/negative expression of CD45RA andexpression of CD27 and CD8 were sorted into α4β7 negative, intermediateand high populations using Act1-phycoerythrin. For CD4 lymphocytes,memory CD4 lymphocytes defined by lack of CD45RA and expression of CD4were sorted into α4β7^(−ve) CLA^(−ve), α4β7^(−ve) CLA^(+ve), andα4β7^(+ve)CLA^(−ve) sub-populations based on CLA and α4β7 expressionusing the anti-α4β7 antibody Act1-Phyorythrin and the anti-CLA antibodyHECA 452-FITC. These sub-populations of memory CD4 (FIG. 13A) and CD8(FIG. 13B) lymphocytes were then examined for their ability to chemotaxto 1 μM TECK (n=2).

FIGS. 14A-14B illustrates a nucleotide sequence encoding human (Homosapiens) GPR-9-6 (SEQ ID NO:1) deposited in Genbank under AccessionNumber U45982, having an open-reading frame beginning at position 58.

FIG. 15 illustrates the amino acid sequence of a human GPR-9-6 protein(SEQ ID NO:2) encoded by the DNA sequence shown in FIGS. 14A-14B (SEQ IDNO:1).

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations used herein include: ECV304, human umbilical veinendothelial cell line (ATCC Accession No. CRL-1998); ADEC, adenoidexpressed chemokine; IP10, IFN-gamma-inducible 10 kDa protein; IMDC,immature dendritic cell; I-TAC, interferon-inducible T cell alphachemoattractant; MCP-1, monocyte chemoattractant protein; SDF, stromalcell derived factor; MDC, mature dendritic cell chemokine; MIG, monokineinduced by interferon-gamma; RANTES, regulated on activation normal Tcell expressed; MIP3, macrophage inflammatory protein 3; MIP4,macrophage inflammatory protein 4; TECK, thymus expressed chemokine;SLC, secondary lymphoid-tissue chemokine; DC, dendritic cell.

Chemokines and their receptors constitute an important component in theregulation of directed leukocyte migration. Chemokines are produced atsites of inflammation and attract various leukocytes bearing thecorresponding receptors. While the spectrum of chemokines expressed atthe inflammatory site can differentially attract certain inflammatorycells, selectivity and variation in chemokine receptor expression onleukocytes provides further regulation to ensure appropriate cellrecruitment in response to particular inflammatory stimuli. As thenumber of identified and characterized chemokine receptors continues togrow, it is becoming increasingly clear that cells selectively expressseveral receptors which may identify, mark, or otherwise characterizefunctional subsets of leukocytes such as T_(H)1 and T_(H)2, naive andmemory, activated and quiescent T cells. Because several characterizedand/or orphan chemokine receptors can be co-expressed on individualcells, it has been difficult to validate the role of specific receptorsin the initiation and progression of disease or, for that matter, innormal immune function.

As described herein, a study of the orphan chemokine receptor GPR-9-6was conducted. In the course of the study an antibody which binds humanGPR-9-6 (mAb 3C3) was produced and used to analyze the expression andfunction of the receptor on various types of leukocytes. The receptorwas found to be expressed predominantly on thymocytes and α4β7^(hi) CD4⁺memory lymphocytes which home to mucosal sites (e.g., respiratory tract,urogenital tract, alimentary canal and associated tissues (pancreas,gallbladder). As described herein, GPR-9-6 is a functional CC chemokinereceptor which binds and is activated by the CC chemokine known asthymus-expressed chemokine (TECK).

The invention relates to the chemokine receptor GPR-9-6 and to agents(e.g., ligands, antibodies, antagonists, agonists) which bind to thereceptor. In one aspect, the invention relates to an antibody whichbinds to mammalian GPR-9-6 or a portion of GPR-9-6.

Antibodies and Antibody Producing Cells

The antibody of the invention can be polyclonal or monoclonal, and theterm “antibody” is intended to encompass both polyclonal and monoclonalantibodies. The terms polyclonal and monoclonal refer to the degree ofhomogeneity of an antibody preparation, and are not intended to belimited to particular methods of production. The term “antibody” as usedherein also encompasses functional fragments of antibodies, includingfragments of chimeric, humanized, primatized, veneered or single chainantibodies. Functional fragments include antigen-binding fragments whichbind to a mammalian GPR-9-6. For example, antibody fragments capable ofbinding to a mammalian GPR-9-6 or portions thereof, including, but notlimited to Fv, Fab, Fab′ and F(ab′)₂ fragments are encompassed by theinvention. Such fragments can be produced by enzymatic cleavage or byrecombinant techniques. For example, papain or pepsin cleavage cangenerate Fab or F(ab′)₂ fragments, respectively. Other proteases withthe requisite substrate specificity can also be used to generate Fab orF(ab′)₂ fragments. Antibodies can also be produced in a variety oftruncated forms using antibody genes in which one or more stop codonshas been introduced upstream of the natural stop site. For example, achimeric gene encoding a F(ab′)₂ heavy chain portion can be designed toinclude DNA sequences encoding the CH₁ domain and hinge region of theheavy chain.

Single chain antibodies, and chimeric, humanized or primatized(CDR-grafted), or veneered antibodies, as well as chimeric, CDR-graftedor veneered single chain antibodies, comprising portions derived fromdifferent species, and the like are also encompassed by the presentinvention and the term “antibody”. The various portions of theseantibodies can be joined together chemically by conventional techniques,or can be prepared as a contiguous protein using genetic engineeringtechniques. For example, nucleic acids encoding a chimeric or humanizedchain can be expressed to produce a contiguous protein. See, e.g.,Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European PatentNo. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533;Neuberger, M. S. et al., European Patent No. 0,194,276 B 1; Winter, U.S.Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Queen etal., European Patent No. 0 451 216 B1; and Padlan, E. A. et al., EP 0519 596 A1. See also, Newman, R. et al., BioTechnology, 10: 1455-1460(1992), regarding primatized antibody, and Ladner et al., U.S. Pat. No.4,946,778 and Bird, R. E. et al., Science, 242: 423-426 (1988))regarding single chain antibodies.

Humanized antibodies can be produced using synthetic or recombinant DNAtechnology using standard methods or other suitable techniques. Nucleicacid (e.g., cDNA) sequences coding for humanized variable regions canalso be constructed using PCR mutagenesis methods to alter DNA sequencesencoding a human or humanized chain, such as a DNA template from apreviously humanized variable region (see e.g., Kamman, M., et al.,Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research,53: 851-856 (1993); Daugherty, B. L. et al., Nucleic Acids Res., 19(9):2471-2476 (1991); and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302(1991)). Using these or other suitable methods, variants can also bereadily produced. In one embodiment, cloned variable regions can bemutated, and sequences encoding variants with the desired specificitycan be selected (e.g., from a phage library; see e.g., Krebber et al.,U.S. Pat. No. 5,514,548; Hoogenboom et al., WO 93/06213, published Apr.1, 1993).

Antibodies which are specific for mammalian (e.g., human) GPR-9-6 can beraised against an appropriate immunogen, such as isolated and/orrecombinant human GPR-9-6 or portions thereof (including syntheticmolecules, such as synthetic peptides). Antibodies can also be raised byimmunizing a suitable host (e.g., mouse) with cells that expressGPR-9-6, such as thymocytes. In addition, cells expressing a recombinantmammalian GPR-9-6 such as transfected cells, can be used as immunogensor in a screen for antibody which binds receptor (See e.g., Chuntharapaiet al., J. Immunol., 152: 1783-1789 (1994); Chuntharapai et al., U.S.Pat. No. 5,440,021).

Preparation of immunizing antigen, and polyclonal and monoclonalantibody production can be preformed using any suitable technique. Avariety of methods have been described (see e.g., Kohler et al., Nature,256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein etal., Nature 266: 550-552 (1977); Koprowski et al., U.S. Pat. No.4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.);Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer'94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.),Chapter 11, (1991)). Generally, a hybridoma is produced by fusing asuitable immortal cell line (e.g., a myeloma cell line such as SP2/0,P3X63Ag8.653 or a heteromyloma) with antibody producing cells. Antibodyproducing cells can be obtained from the peripheral blood or, preferablythe spleen or lymph nodes, of humans or other suitable animals immunizedwith the antigen of interest. The fused cells (hybridomas) can beisolated using selective culture conditions, and cloned by limitingdilution. Cells which produce antibodies with the desired specificitycan be selected by a suitable assay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies of therequisite specificity (e.g., human antibodies or antigen-bindingfragments) can be used, including, for example, methods which selectrecombinant antibody from a library (e.g., a phage display library), orwhich rely upon immunization of transgenic animals (e.g., mice) capableof producing a repertoire of human antibodies (see e.g., Jakobovits etal., Proc. Natl. Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits etal., Nature, 362: 255-258 (1993); Lonberg et al., U.S. Pat. No.5,545,806; Surani et al., U.S. Pat. No. 5,545,807; Lonberg et al.,WO97/13852).

In one embodiment, the antibody or antigen-binding fragment thereof hasspecificity for a mammalian GPR-9-6, preferably a naturally occurring orendogenous human GPR-9-6. In another embodiment, the antibody is an IgGor antigen-binding fragment of an IgG. In another embodiment, theantibody or antigen-binding fragment can bind to a mammalian GPR-9-6 andinhibit (reduce or prevent) one or more functions of the receptor. In apreferred embodiment, the antibody or antigen-binding fragment caninhibit binding of a ligand (i.e., one or more ligands) to the receptor,and/or one or more functions mediated by GPR-9-6 in response to ligandbinding.

In a particular embodiment, the antibody or antigen-binding fragment caninhibit the binding of a mammalian (e.g., human) TECK to mammalian(e.g., human) GPR-9-6 and/or one or more functions mediated by GPR-9-6in respond to TECK binding. In a particularly preferred embodiment, theantibody or antigen-binding fragment can inhibit the binding of TECK toGPR-9-6 and, thereby inhibit TECK-induced chemotaxis.

As shown herein, TECK is a ligand for GPR-9-6 and activates the receptorleading to TECK-induced Ca²⁺ flux in cells that express GPR-9-6 (FIG.9A). Cells that express mammalian GPR-9-6, including recombinant cells,can also undergo TECK-induced chemotaxis (FIGS. 8A-8D, 8F, 10, 11A-11Band 13A-13B). Other functions which can be mediated by GPR-9-6 inresponse to ligand binding (e.g., TECK) include, for example, signaltransduction (e.g., GDP/GTP exchange by GPR-9-6 associated G proteins,transient increase in the concentration of cytosolic free calcium[Ca²⁺]_(i)) and GPR-9-6-mediated processes and cellular responses (e.g.,proliferation, migration, chemotaxis, secretion, degranulation,inflammatory mediator release (such as release of bioactive lipids suchas leukotrienes (e.g., leukotriene C₄)), respiratory burst).

In another embodiment, the binding of the antibody or antigen-bindingfragment thereof to mammalian (e.g., human) GPR-9-6 can be inhibited bya peptide that consists of the amino acid sequence of SEQ ID NO:3.

As described herein, an antibody designated “mAb 3C3” that binds humanGPR-9-6 has been produced. mAb 3C3 can be produced by murine hybridoma3C3, also referred to as murine hybridoma LS129-3C3-E3-1 which wasdeposited on Mar. 4, 1999, on behalf of LeukoSite, Inc., 215 FirstStreet, Cambridge, Mass. 02142, U.S.A., now Millennium Pharmaceuticals,Inc., 75 Sidney Street, Cambridge, Mass. 01239, at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, U.S.A., under Accession No. HB-12653. In another embodiment,the anti-GPR-9-6 antibody of the invention is mAb 3C3 or anantigen-binding fragment thereof. In another embodiment, the binding ofthe antibody or antigen-binding fragment to mammalian (e.g., human)GPR-9-6 can be inhibited by mAb 3C3. Such inhibition can be the resultof competition for the same or similar epitope, steric interference ordue to a change in the conformation of GPR-9-6 that is induced uponantibody binding to the receptor. In still another embodiment, theantibody or antigen-binding fragment of the invention has the same orsimilar epitopic specificity as mAb 3C3. Antibodies with an epitopicspecificity which is the same as or similar to that of mAb 3C3 can beidentified by a variety of suitable methods. For example, an antibodywith the same or similar epitopic specificity as mAb 3C3 can beidentified based upon the ability to compete with mAb 3C3 for binding tomammalian GPR-9-6. In another example, the binding of mAb 3C3 and thebinding of an antibody with the same or similar epitopic specificity tomammalian GPR-9-6 can be inhibited by a single peptide (e.g., naturalpeptide, synthetic peptide). The peptide can comprise nine to aboutfifty amino acids. Preferably, the peptide comprises nine to abouttwenty-six amino acids. In still another example, an antibody with thesame or similar epitopic specificity as mAb 3C3 can be identified usingchimeric receptors (see e.g., Rucker et al., Cell 87:437-446 (1996)).

The invention also relates to a bispecific antibody, or functionalfragment thereof (e.g., F(ab′)₂), which binds to a mammalian GPR-9-6 andat least one other antigen. In a particular embodiment, the bispecificantibody, or functional fragment thereof has the same or similarepitopic specificity as mAb 3C3 and at least one other antibody (see,e.g., U.S. Pat. No. 5,141,736 (Iwasa et al.), U.S. Pat. Nos. 4,444,878,5,292,668, 5,523,210 (all to Paulus et al.) and U.S. Pat. No. 5,496,549(Yamazaki et al.)).

In a preferred embodiment, the antibody or antigen-binding fragment ofthe invention specifically binds to a mammalian GPR-9-6. As used hereinthe term “specific antibody” or “specific” when referring to anantibody-antigen interaction is used to indicate that the antibody canselectively bind to a mammalian GPR-9-6, rather than to indicate thatthe antibody can bind to only one antigen. For example, an antibody maybind to one or several antigens with low affinity and bind to humanGPR-9-6 with a high affinity. Such an antibody is considered to bespecific for human GPR-9-6 because when used (e.g., in therapeutic ordiagnostic application) at a suitable concentration, the antibody canselectively bind to human GPR-9-6. The concentration of antibodyrequired to provide selectivity for a mammalian GPR-9-6 (e.g., aconcentration which reduces or eliminates low affinity binding) can bereadily determined by suitable methods, for example, titration.

In another aspect, the invention relates to an isolated cell whichproduces an antibody or an antigen-binding fragment of an antibody thatbinds to a mammalian GPR-9-6. In a preferred embodiment, the isolatedantibody-producing cell of the invention is an immortalized cell, suchas a hybridoma, heterohybridoma, lymphoblastoid cell or a recombinantcell. The antibody-producing cells of the present invention have usesother than for the production of antibodies. For example, the cell ofthe present invention can be fused with other cells (such as suitablydrug-marked human myeloma, mouse myeloma, human-mouse heteromyeloma orhuman lymphoblastoid cells) to produce, for example, additionalhybridomas, and thus provide for the transfer of the genes encoding theantibody. In addition, the cell can be used as a source of nucleic acidsencoding the anti-GPR-9-6 immunoglobulin chains, which can be isolatedand expressed (e.g., upon transfer to other cells using any suitabletechnique (see e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Winter,U.S. Pat. No. 5,225,539)). For instance, clones comprising a sequenceencoding a rearranged anti-GPR-9-6 light and/or heavy chain can beisolated (e.g., by PCR) or cDNA libraries can be prepared from mRNAisolated from the cell lines, and cDNA clones encoding an anti-GPR-9-6immunoglobulin chain(s) can be isolated. Thus, nucleic acids encodingthe heavy and/or light chains of the antibodies or portions thereof canbe obtained and used for the production of the specific immunoglobulin,immunoglobulin chain, or variants thereof (e.g., humanizedimmunoglobulin) in a variety of host cells or in an in vitro translationsystem. For example, the nucleic acids, including cDNAs, or derivativesthereof encoding variants such as a humanized immunoglobulin orimmunoglobulin chain, can be placed into suitable prokaryotic oreukaryotic vectors (e.g., expression vectors) and introduced into asuitable host cell by an appropriate method (e.g., transformation,transfection, electroporation, infection), such that the nucleic acid isoperably linked to one or more expression control elements (e.g., in thevector or integrated into the host cell genome), to produce arecombinant antibody-producing cell.

The antibody of the invention can be produced by any suitable method,for example, by collecting serum from an animal (e.g., mouse, human,transgenic mouse) which has been immunized with a mammalian GPR-9-6. Inanother example, a suitable antibody producing cell (e.g., hybridoma,heterohybridoma, lymphoblastoid cell, recombinant cell) can bemaintained, either in vitro or in vivo, under conditions suitable forexpression (e.g., in the presence of inducer, suitable mediasupplemented with appropriate salts, growth factors, antibiotic,nutritional supplements), whereby the antibody or antigen-bindingfragment is produced. If desired, the antibody or antigen-bindingfragment can be recovered and/or isolated (e.g., from the host cells,culture medium) and purified to the desired degree. Recovery andpurification of the antibody can be achieved using suitable methods,such as, centrifugation, filtration, column chromatography (e.g.,ion-exchange, gel filtration, hydrophobic-interaction, affinity),preparative native electrophoresis, precipitation and ultrafiltration.It will be appreciated that the method of production encompassesexpression in a host cell of a transgenic animal (see e.g., WO 92/03918,GenPharm International, published Mar. 19, 1992).

As described herein, antibodies and functional fragments thereof of thepresent invention can inhibit (reduce or prevent) binding of a ligand toa mammalian GPR-9-6 and/or inhibit one or more functions associated withbinding of the ligand to GPR-9-6. As discussed below various methods canbe used to assess inhibition of binding of a ligand to GPR-9-6 and/orfunction associated with binding of the ligand to the receptor.

Binding Assays

The invention also relates to methods for detecting or identifying anagent (i.e., molecule or compound) which can bind to a mammalian GPR-9-6or a ligand-binding variant thereof.

As used herein “mammalian GPR-9-6” refers to naturally occurring orendogenous mammalian GPR-9-6 proteins and to proteins having an aminoacid sequence which is the same as that of a naturally occurring orendogenous corresponding mammalian GPR-9-6 protein (e.g., recombinantproteins, synthetic proteins (i.e., produced using the methods ofsynthetic organic chemistry)). Accordingly, as defined herein, the termincludes mature receptor protein, polymorphic or allelic variants, andother isoforms of a mammalian GPR-9-6 (e.g., produced by alternativesplicing or other cellular processes), and modified or unmodified formsof the foregoing (e.g., lipidated, glycosylated, unglycosylated).Naturally occurring or endogenous mammalian GPR-9-6 proteins includewild type proteins such as mature GPR-9-6, polymorphic or allelicvariants and other isoforms which occur naturally in mammals (e.g.,humans, non-human primates). Such proteins can be recovered or isolatedfrom a source which naturally produces mammalian GPR-9-6, for example.These proteins and mammalian GPR-9-6 proteins having the same amino acidsequence as a naturally occurring or endogenous corresponding mammalianGPR-9-6, are referred to by the name of the corresponding mammal. Forexample, where the corresponding mammal is a human, the protein isdesignated as a human GPR-9-6 protein (e.g., a recombinant human GPR-9-6produced in a suitable host cell).

“Functional variants” of mammalian GPR-9-6 proteins include functionalfragments, functional mutant proteins, and/or functional fusion proteinswhich can be produce using suitable methods (e.g., mutagenesis (e.g.,chemical mutagenesis, radiation mutagenesis), recombinant DNAtechniques). A “functional variant” is a protein or polypeptide whichhas at least one function characteristic of a mammalian GPR-9-6 proteinas described herein, such as a binding activity, a signaling activityand/or ability to stimulate a cellular response. Preferred functionalvariants can bind a ligand (i.e., one or more ligands, such as TECK).

Generally, fragments or portions of mammalian GPR-9-6 proteins includethose having a deletion (i.e., one or more deletions) of an amino acid(i.e., one or more amino acids) relative to the mature mammalian GPR-9-6protein (such as N-terminal, C-terminal or internal deletions).Fragments or portions in which only contiguous amino acids have beendeleted or in which non-contiguous amino acids have been deletedrelative to mature mammalian GPR-9-6 protein are also envisioned.

Mutant mammalian GPR-9-6 proteins include natural or artificial variantsof a mammalian GPR-9-6 protein differing by the addition, deletionand/or substitution of one or more contiguous or non-contiguous aminoacid residues (e.g., receptor chimeras). Such mutations can occur at oneor more sites on a protein, for example a conserved region ornonconserved region (compared to other chemokine receptors or G-proteincoupled receptors), extracellular region, cytoplasmic region, ortransmembrane region.

Fusion proteins encompass polypeptides comprising a mammalian GPR-9-6(e.g., human GPR-9-6) or a valiant thereof as a first moiety, linked viaa covalent bond (e.g., a peptide bond) to a second moiety not occurringin the mammalian GPR-9-6 as found in nature. Thus, the second moiety canbe an amino acid, oligopeptide or polypeptide. The second moiety can belinked to the first moiety at a suitable position, for example, theN-terminus, the C-terminus or internally. In one embodiment, the fusionprotein comprises an affinity ligand (e.g., an enzyme, an antigen,epitope tag, a binding domain) as the first moiety, and a second moietycomprising a linker sequence and human GPR-9-6 or a portion thereof.Additional (e.g., third, fourth) moieties can be present as appropriate.

In one embodiment, a functional variant of mammalian GPR-9-6 (e.g., aligand binding variant) shares at least about 80% amino acid sequencesimilarity with said mammalian GPR-9-6, preferably at least about 90%amino acid sequence similarity, and more preferably at least about 95%amino acid sequence similarity with said mammalian GPR-9-6. In anotherembodiment, a functional fusion protein comprises a first moiety whichshares at least about 85% sequence similarity with a mammalian GPR-9-6,preferably at least about 90% sequence similarity, and more preferablyat least about 95% sequence similarity with a mammalian GPR-9-6 (e.g., ahuman GPR9-6 (e.g., SEQ ID NO:2)). In another embodiment, a functionalmammalian GPR-9-6 protein or functional variant of a mammalian GPR-9-6protein shares at least about 80% amino acid sequence similarity,preferably at least about 90% amino acid sequence similarity, and morepreferably at least about 95% amino acid sequence similarity with anaturally occurring human GPR-9-6 (e.g., SEQ ID NO:2). Amino acidsequence similarity can be determined using a suitable sequencealignment algorithm, such as the LASERGENE system (sequence assembly andalignment software; DNASTAR, Inc., Madison, Wis.), using the Clustalmethod with the PAM 250 residue weight table, a gap penalty of 10, a gaplength penalty of 10 and default parameters (pairwise alignmentparameters: ktuple=1, gap penalty=3, window=4 and diagonals saved=5). Inanother embodiment, a functional variant is encoded by a nucleic acidsequence which is different from the naturally-occurring nucleic acidsequence, but which, due to the degeneracy of the genetic code, encodesmammalian GPR-9-6 or a portion thereof.

A composition comprising a mammalian GPR-9-6 or functional variantthereof can be used in a binding assay to detect and/or identify agentsthat can bind to the receptor. Compositions suitable for use in abinding assay include, for example, cells which naturally express amammalian GPR-9-6 or functional variant thereof (e.g., thymocytes,GPR-9-6⁺ CLA^(−ve) α4β7^(hi) CD4⁺ memory lymphocytes, cell lines (e.g.,MOLT-4 (ATCC Accession No. CRL-1582), MOLT-13 (M. Brenner, Brigham andWomans Hospital, Boston, Mass.)) and recombinant cells comprising anexogenous nucleic acid sequence which encodes a mammalian GPR-9-6 orfunctional variant thereof. Compositions suitable for use in a bindingassay also include, membrane preparations which comprise a mammalianGPR-9-6 or functional variant thereof. Such membrane preparations cancontain natural (e.g., plasma membrane) or synthetic membranes.Preferably, the membrane preparation is a membrane fraction of a cellthat expresses a mammalian GPR-9-6 or a functional variant thereof.

In one embodiment, the method of detecting or identifying an agent thatbinds to a mammalian GPR-9-6 is a competitive binding assay in which theability of a test agent to inhibit the binding of a reference agent(e.g., a ligand, an antibody) is assessed. For example, the referenceagent can be labeled with a suitable label as described herein, and theamount of labeled reference agent required to saturate the GPR-9-6present in the assay can be determined. A saturating amount of labeledreference agent and various amounts of a test agent can be contactedwith a composition comprising a mammalian GPR-9-6 or functional variantthereof under conditions suitable for binding and complex formationdetermined.

The formation of a complex between the reference agent and the GPR-9-6or functional variant thereof can be detected or measured directly orindirectly using suitable methods. For example, the agent can be labeledwith a suitable label and the formation of a complex can be determinedby detection of the label. The specificity of the complex can bedetermined using a suitable control such as unlabeled agent or labelalone. Labels suitable for use in detection of a complex between anagent and a mammalian GPR-9-6 or functional variant thereof include, forexample, a radioisotope, an epitope, an affinity label (e.g., biotin,avidin), a spin label, an enzyme, a fluorescent group or achemiluminescent group.

The capacity of the test agent to inhibit the formation of a complexbetween the reference agent and a mammalian GPR-9-6 can be reported asthe concentration of test agent required for 50% inhibition (IC₅₀values) of specific binding of labeled reference agent. Specific bindingis preferably defined as the total binding (e.g., total label incomplex) minus the non-specific binding. Non-specific binding ispreferably defined as the amount of label still detected in complexesformed in the presence of excess unlabeled reference agent. Referenceagents which are suitable for use in the method include molecules andcompounds which specifically bind to a mammalian GPR-9-6 or a functionalvariant thereof, for example, a ligand of GPR-9-6 or an antibody. In apreferred embodiment, the reference agent is mAb 3C3. In a particularlypreferred embodiment, the reference agent is a mammalian (e.g., human)TECK.

The invention also relates to a method of identifying or isolating anagent (i.e., molecule or compound) which can be used in therapy, asdescribed herein. In one embodiment, the agent is identified or isolatedin a competitive binding assay as described above. In anotherembodiment, cells which express a mammalian GPR-9-6 or a functionalvariant thereof are maintained under conditions appropriate forexpression of receptor. The cells are contacted with an agent (e.g.,ligand, antagonist, agonist) under conditions suitable for binding(e.g., in a suitable binding buffer), and the formation of a complexbetween the agent and a mammalian GPR-9-6 is detected or measured usingsuitable techniques. For example, the agent can be labeled as describedherein and the amount of label present in an agent-GPR-9-6 complex canbe determined. The extent of complex formation can be determinedrelative to a suitable control (e.g., compared with backgrounddetermined in the absence of agent, compared with binding of a secondagent (i.e., a standard, an isotype control), compared with binding ofagent to cells that do not express GPR-9-6).

Thus, the invention relates to a method of identifying or isolating anagent for use in treating a subject having an inflammatory disease. Inparticular embodiments, the method is a method of identifying orisolating an agent for use in treating a subject having an inflammatorydisease associated with mucosal tissue, such as Crohn's disease orcolitis. In another embodiment, the method is a method of identifying orisolating an agent for use in inhibiting GPR-9-6-mediated homing ofleukocytes in a subject. In another embodiment, the method is a methodof identifying or isolating an agent for use in modulating a GPR-9-6function in a subject.

According to the method of the present invention, agents can beindividually screened or one or more agents can be tested simultaneouslyaccording to the methods herein. Where a mixture of compounds is tested,the compounds selected by the processes described can be separated (asappropriate) and identified by suitable methods (e.g., sequencing,chromatography). The presence of one or more compounds (e.g., a ligand,inhibitor, promoter) in a test sample can also be determined accordingto these methods.

Agents which bind to a mammalian GPR-9-6 and which are useful in thetherapeutic methods described herein can be identified, for example, byscreening libraries or collections of molecules, such as, the ChemicalRepository of the National Cancer Institute, in assays described hereinor using other suitable methods. Large combinatorial libraries ofcompounds (e.g., organic compounds, recombinant or synthetic peptides,“peptoids”, nucleic acids) produced by combinatorial chemical synthesisor other methods can be tested (see e.g., Zuckerman, R. N. et al., J.Med. Chem., 37: 2678-2685 (1994) and references cited therein; see also,Ohlmeyer, M. H. J. et al., Proc. Natl. Acad. Sci. USA 90:10922-10926(1993) and DeWitt, S. H. et al., Proc. Natl. Acad. Sci. USA 90:6909-6913(1993), relating to tagged compounds; Rutter, W. J. et al. U.S. Pat. No.5,010,175; Huebner, V. D. et al., U.S. Pat. No. 5,182,366; and Geysen,H. M., U.S. Pat. No. 4,833,092). Where compounds selected from acombinatorial library by the present method carry unique tags,identification of individual compounds by chromatographic methods ispossible. In one embodiment, the collection of agents tested accordingto the method of the invention does not comprise chemokines or mutantsor analogues thereof.

Functional Assays

An agent which binds a mammalian GPR-9-6 or a functional variant thereofcan be further studied in one or more suitable assays to determine ifsaid agent can modulate (inhibit (reduce or prevent) or promote) one ormore functions of GPR-9-6 as described herein. For example, an agent canbe tested in an extracellular acidification assay, calcium flux assay,ligand binding assay, chemotaxis assay or assay which monitorsdegranulation or inflammatory mediator release (see, for example,Hesselgesser et al., J. Biol. Chem. 273(25):15687-15692 (1998) and WO98/02151).

For example, an agent which binds to a mammalian GPR-9-6 can be testedin a leukocyte chemotaxis assay using suitable cells. Suitable cellsinclude, for example, cell lines, recombinant cells or isolated cellswhich express a mammalian GPR-9-6 and undergo GPR-9-6 ligand-induced(e.g., TECK) chemotaxis. In one example, GPR-9-6-expressing recombinantL1.2 cells (see Campbell, et al. J Cell Biol, 134:255-266 (1996)regarding L1.2 cells), can be used in a modification of atransendothelial migration assay (Carr, M. W., et al. T. A., Proc. NatlAcad Sci, USA, (91):3652 (1994)). The endothelial cells used in thisassay are preferably the endothelial cell line, ECV 304, which can beobtained from the American Type Culture Collection (Manassas, Va.).Endothelial cells can be cultured on 6.5 mm diameter Transwell cultureinserts (Costar Corp., Cambridge, Mass.) with 3.0 μm pore size. Culturemedia for the ECV 304 cells can consist of M199+10% FCS, L-glutamine,and antibiotics. The assay media can consist of equal parts RPMI 1640and M199 with 0.5% BSA. Two hours before the assay, 2×10⁵ ECV 304 cellscan be plated onto each insert of the 24 well Transwell chemotaxis plateand incubated at 37° C. Chemotactic factor such as TECK (Peprotech,Rocky Hill, N.J.) (diluted in assay medium) can be added to the 24-welltissue culture plates in a final volume of 600 μL. Endothelial-coatedTranswell can be inserted into each well and 10⁶ cells of the leukocytetype being studied are added to the top chamber in a final volume of 100μL of assay medium. The plate can then be incubated at 37° C. in 5%CO₂/95% air for 1-2 hours. The cells that migrate to the bottom chamberduring incubation can be counted, for example using flow cytometry. Tocount cells by flow cytometry, 500 μL of the cell suspension from thelower chamber can be placed in a tube and relative counts can obtainedfor a set period of time, for example, 30 seconds. This counting methodis highly reproducible and allows gating on the leukocytes and theexclusion of debris or other cell types from the analysis.Alternatively, cells can be counted with a microscope. Assays toevaluate agents that can inhibit or promote chemotaxis can be performedin the same way as control experiment described above, except that agentsolutions, in assay media containing up to 1% of DMSO co-solvent, can beadded to both the top and bottom chambers prior to addition of thecells. The capacity of an agent to inhibit or promote chemotaxis can bedetermined by comparing the number of cell that migrate to the bottomchamber in wells which contain the agent, to the number of cells whichmigrate to the bottom chamber in control wells. Control wells cancontain equivalent amounts of DMSO, but no agent.

An agent which binds to a mammalian GPR-9-6 can also be assessed bymonitoring cellular responses induced by active receptor, using suitablecells which express a mammalian GPR-9-6 or a functional valiant thereof.For instance, exocytosis (e.g., degranulation of cells leading torelease of one or more enzymes or other granule components, such asesterases (e.g., serine esterases), perforin, and/or granzymes),inflammatory mediator release (such as release of bioactive lipids suchas leukotrienes (e.g., leukotriene C₄)), and respiratory burst, can bemonitored by methods known in the art or other suitable methods (seee.g., Taub, D. D. et al., J. Immunol., 155: 3877-3888 (1995), regardingassays for release of granule-derived serine esterases; Loetscher etal., J. Immunol., 156: 322-327 (1996), regarding assays for enzyme andgranzyme release; Rot, A. et al., J. Exp. Med., 176: 1489-1495 (1992)regarding respiratory burst; Bischoff, S. C. et al., Eur. J. Immunol.,23: 761-767 (1993) and Baggliolini, M. and C. A. Dahinden, ImmunologyToday, 15: 127-133 (1994)).

In one embodiment, an agent that can inhibit or promote a function ofGPR-9-6 is identified by monitoring the release of an enzyme upondegranulation or exocytosis by a cell capable of this function. Cellsexpressing a mammalian GPR-9-6 or a functional variant thereof can bemaintained in a suitable medium under suitable conditions, anddegranulation can be induced. The cells are contacted with an agent tobe tested, and enzyme release can be assessed. The release of an enzymeinto the medium can be detected or measured using a suitable assay, suchas an immunological assay, or biochemical assay for enzyme activity.

The medium can be assayed directly, by introducing components of theassay (e.g., substrate, co-factors, antibody) into the medium (e.g.,before, simultaneous with or after the cells and agent are combined).The assay can also be performed on medium which has been separated fromthe cells or further processed (e.g., fractionated) prior to assay. Forexample, convenient assays are available for enzymes, such as serineesterases (see e.g., Taub, D. D. et al., J. Immunol., 155: 3877-3888(1995) regarding release of granule-derived serine esterases).

In another embodiment, cells expressing a mammalian GPR-9-6 or afunctional variant thereof are combined with a ligand of GPR-9-6 (e.g.,TECK), an agent to be tested is added before, after or simultaneoustherewith, and Ca²⁺ flux is assessed. Inhibition of ligand-induced Ca²⁺flux is indicative that the agent is an inhibitor or antagonist ofmammalian GPR-9-6 function.

Cellular adherence can be monitored by methods known in the art or othersuitable methods. Engagement of the chemokine receptors of a lymphocytecan cause integrin activation, and induction of adherence to adhesionmolecules expressed in vasculature or the perivascular space. In oneembodiment, a ligand, inhibitor and/or promoter of GPR-9-6 function isidentified by monitoring cellular adherence by a cell capable ofadhesion. For example, an agent to be tested can be combined with (a)cells expressing a mammalian GPR-9-6 or a functional variant thereof(preferably non-adherent cells which when transfected with receptoracquire adhesive ability), (b) a composition comprising a suitableadhesion molecule (e.g., a substrate such as a culture well coated withan adhesion molecule, such as fibronectin), and (c) a ligand or promoter(e.g., agonist), and maintained under conditions suitable for ligand- orpromoter-induced adhesion. Labeling of cells with a fluorescent dyeprovides a convenient means of detecting adherent cells. Nonadherentcells can be removed (e.g., by washing) and the number of adherent cellsdetermined. The effect of the agent in inhibiting or enhancing ligand-or promoter-induced adhesion can be indicative of inhibitor or promoteractivity, respectively. Agents active in the assay include inhibitorsand promoters of binding, signaling, and/or cellular responses. Inanother embodiment, an agent to be tested can be combined with cellsexpressing a mammalian GPR-9-6 and a composition comprising a suitableadhesion molecule under conditions suitable for ligand- orpromoter-induced adhesion, and adhesion is monitored. Increased adhesionrelative to a suitable control is indicative of the presence of a ligandand/or promoter.

The binding assays and functional assays described above can be used,alone or in combination with each other or other suitable methods, todetect or identify agents which bind a mammalian GPR-9-6 protein and/ormodulators (inhibitors, promoters) of a GPR-9-6 protein function. The invitro methods of the present invention can be adapted forhigh-throughput screening in which large numbers of samples areprocessed (e.g., a 96-well format). Cells expressing a mammalian GPR-9-6(e.g., human GPR-9-6) or a functional variant thereof at levels suitablefor high-throughput screening can be used, and thus, are particularlyvaluable in the identification and/or isolation of agents which bindGPR-9-6, and modulators of GPR-9-6 function. Expression of GPR-9-6 canbe monitored in a variety of ways. For instance, expression can bemonitored using antibodies of the present invention which bind receptoror a portion thereof. Also, commercially available antibodies can beused to detect expression of an antigen- or epitope-tagged fusionprotein comprising a receptor protein or polypeptide (e.g., FLAG taggedreceptors), and cells expressing the GPR-9-6 at the desired level can beselected (e.g., by flow cytometry).

Models of Inflammation

In vivo models of inflammation are available which can be used to assessthe efficacy of antibodies and antigen-binding fragments of theinvention as well as agents identified by the methods described hereinas in vivo as therapeutics. For example, leukocyte infiltration uponintradermal injection of a chemokine and an antibody or fragment thereofreactive with mammalian GPR-9-6 into a suitable animal, such as rabbit,mouse, rat, guinea pig or primate (e.g., rhesus macaque) can bemonitored (see e.g., Van Damme, J. et al., J. Exp. Med., 176: 59-65(1992); Zachariae, C.O.C. et al., J. Exp. Med. 171: 2177-2182 (1990);Jose, P. J. et al., J. Exp. Med. 179: 881-887 (1994)). In oneembodiment, skin biopsies are assessed histologically for infiltrationof leukocytes (e.g., GPR-9-6⁺ T cells). In another embodiment, labeledcells (e.g., stably transfected cells expressing a mammalian GPR-9-6,labeled with Hun for example) capable of chemotaxis and extravasationare administered to the animal. For example, an antibody or agent to beassessed which binds a mammalian GPR-9-6 can be administered, eitherbefore, simultaneously with or after a GPR-9-6 ligand or agonist (e.g.,TECK) is administered to the test animal. A decrease of the extent ofinfiltration in the presence of antibody or agent as compared with theextent of infiltration in the absence of said antibody or agent isindicative of inhibition.

As described herein, GPR-9-6 is selectively expressed on memorylymphocytes which home to mucosal sites (e.g., CLA^(−ve) α4β7^(hi) CD4⁺lymphocytes). Thus, animal models of inflammatory diseases of the mucosa(e.g., respiratory tract, urogenital tract, alimentary canal andassociated organs and tissues (e.g., pancreas, liver, gall bladder)) canbe used to assess the therapeutic efficacy of GPR-9-6 modulating agents.For example, the antibodies and antigen binding fragments of theinvention as well as agents identified by the methods described hereincan be studied in the cotton-top tamarin model of inflammatory boweldisease (Podolsky, D. K., et al., J. Clin. Invest. 92:372-380 (1993)).The CD45RB^(Hi)/SCID model provides a mouse model with similarity toboth Crohn's disease and ulcerative colitis (Powrie, F. et al.,Immunity, 1: 553-562 (1994)). Therapeutic efficacy in this model can beassessed, for example, by using parameters such as inhibition ofrecruitment of ¹¹¹In-labeled cells to the colon and reduction in thenumber of CD4⁺ T lymphocytes in the lamina propria of the largeintestine after administration (e.g., intravenous (i.v.),intraperitoneally (i.p.) and per oral (p.o.)) of an agent. Knockout micewhich develop intestinal lesions similar to those of human inflammatorybowel disease have also been described (Strober, W. and Ehrhardt, R. O.,Cell, 75: 203-205 (1993)), and NOD mice provide an animal model ofinsulin-dependent diabetes mellitus.

Diagnostic Applications

The antibodies of the present invention have application in proceduresin which GPR-9-6 can be detected on the surface of cells. The receptorprovides a marker of the leukocyte cell types in which it is expressed.For example, antibodies raised against a mammalian GPR-9-6 protein orpeptide, such as the antibodies described herein (e.g., mAb 3C3), can beused to detect and/or quantify cells expressing a mammalian GPR-9-6. Inone embodiment, the antibodies can be used to sort cells which expressGPR-9-6 from among a mixture of cells (e.g., to isolate leukocytes whichhome to the mucosa, such as GPR-9-6⁺ CLA^(−ve) α4β7^(+ve) CD4⁺ memory Tcells). Suitable methods for counting and/or sorting cells can be usedfor this purpose (e.g., flow cytometry, fluorescence activated cellsorting). Cell counts can be used in the diagnosis of diseases orconditions in which an increase or decrease in leukocyte cell types(e.g., leukocytes which home to the mucosa) is observed.

Furthermore, the antibodies can be used to detect or measure expressionof GPR-9-6. For example, antibodies of the present invention can be usedto detect or measure a mammalian GPR-9-6 in a biological sample (e.g.,cells, tissues or body fluids from an individual such as blood, serum,leukocytes (e.g., activated T lymphocytes), bronchoalveolar lavagefluid, saliva, bowel fluid, biopsy specimens). For example, a sample(e.g., tissue and/or fluid) can be obtained from an individual and asuitable assay can be used to assess the presence or amount of GPR-9-6protein. Suitable assays include immunological and immunochemicalmethods such as flow cytometry (e.g., FACS analysis) and enzyme-linkedimmunosorbent assays (ELISA), including chemiluminescence assays,radioimmunoassay, immuno-blot (e.g., western blot) and immunohistology.Generally, a sample and antibody of the present invention are combinedunder conditions suitable for the formation of an antibody-GPR-9-6complex, and the formation of antibody-receptor complex is assessed(directly or indirectly).

The presence of an increased level of GPR-9-6 reactivity in a sample(e.g., a tissue sample) obtained from an individual can be indicative ofinflammation and/or leukocyte (e.g., activated T cell) infiltrationand/or accumulation associated with an inflammatory disease orcondition, such as an inflammatory bowel disease, allograft rejection,delayed type hypersensitivity reaction, or an infection such as a viralor bacterial infection. The presence of a decreased level of GPR-9-6reactivity in the circulation (e.g., on the surface of circulatinglymphocytes) can also be indicative of leukocyte infiltration and/oraccumulation at inflammatory sites. The level of expression of amammalian GPR-9-6 protein or variant can also be used to correlateincreased or decreased expression of a mammalian GPR-9-6 protein with aparticular disease or condition, and in the diagnosis of a disease orcondition in which increased or decreased expression of a mammalianGPR-9-6 protein occurs (e.g., increased or decreased relative to asuitable control, such as the level of expression in a normalindividual). Similarly, the course of therapy can be monitored byassessing GPR-9-6 immunoreactivity in a sample from a subject. Forexample, antibodies of the present invention can be used to monitor thenumber of cells expressing GPR-9-6 in a sample (e.g., blood, tissue)from a subject being treated with an anti-inflammatory orimmunosuppressive agent.

Kits for use in detecting the presence of a mammalian GPR-9-6 protein ina biological sample can also be prepared. Such kits can include anantibody or functional fragment thereof which binds to a mammalianGPR-9-6 receptor or portion of said receptor, as well as one or moreancillary reagents suitable for detecting the presence of a complexbetween the antibody or fragment and GPR-9-6 or portion thereof. Theantibody compositions of the present invention can be provided inlyophilized form, either alone or in combination with additionalantibodies specific for other epitopes. The antibodies, which can belabeled or unlabeled, can be included in the kits with adjunctingredients (e.g., buffers, such as Tris, phosphate and carbonate,stabilizers, excipients, biocides and/or inert proteins, e.g., bovineserum albumin). For example, the antibodies can be provided as alyophilized mixture with the adjunct ingredients, or the adjunctingredients can be separately provided for combination by the user.Generally these adjunct materials will be present in less than about 5%by weight based on the amount of active antibody, and usually will bepresent in a total amount of at least about 0.001% by weight based onantibody concentration. Where a second antibody capable of binding tothe anti-GPR-9-6 antibody is employed, such antibody can be provided inthe kit, for instance in a separate vial or container. The secondantibody, if present, is typically labeled, and can be formulated in ananalogous manner with the antibody formulations described above. Thecomponents (e.g., anti-GPR-9-6 antibody or antigen-binding fragmentthereof, ancillary reagent) of the kit can be packaged separately ortogether within suitable containment means (e.g., bottle, box, envelope,tube). When the kit comprises a plurality of individually packagedcomponents, the individual packages can be contained within a singlelarger containment means (e.g., bottle, box, envelope, tube).

Similarly, the present invention also relates to a method of detectingand/or quantifying expression of a mammalian GPR-9-6 receptor or aportion of the receptor by a cell, in which a composition comprising acell or fraction thereof (e.g., membrane fraction) is contacted with anantibody or functional fragment thereof (e.g., mAb 3C3) which binds to amammalian GPR-9-6 or portion of the receptor under conditionsappropriate for binding of the antibody or fragment thereto, and bindingis monitored. Detection of the antibody, indicative of the formation ofa complex between antibody and a mammalian GPR-9-6 or a portion thereof,indicates the presence of the receptor. Binding of antibody to the cellcan be determined using any suitable method. The method can be used todetect expression of GPR-9-6 on cells from a subject (e.g., in a sample,such as a body fluid, such as blood, saliva or other suitable sample).The level of expression of GPR-9-6 on the surface of cells (e.g.,leukocytes) can also be determined, for instance, by flow cytometry, andthe level of expression (e.g., staining intensity) can be correlatedwith disease susceptibility, progression or risk.

Methods of Therapy

Modulation of mammalian GPR-9-6 function according to the presentinvention, through the inhibition or promotion of at least one functioncharacteristic of a mammalian GPR-9-6 protein, provides an effective andselective way of inhibiting or promoting receptor-mediated functions.Once lymphocytes are recruited to a site, other leukocyte types, such asmonocytes, may be recruited by secondary signals. Thus, agents which canmodulate GPR-9-6 function, including ligands, inhibitors and/orpromoters, such as those identified as described herein, can be used tomodulate leukocyte function (e.g., leukocyte infiltration includingrecruitment and/or accumulation).

In one aspect, the present invention provides a method of modulating(inhibiting or promoting) an inflammatory response in a subject in needof such therapy, comprising administering an effective amount of anagent which inhibits or promotes mammalian GPR-9-6 function to anindividual in need of such therapy. In one embodiment, an effectiveamount of an agent which inhibits one or more functions of a mammalianGPR-9-6 protein (e.g., a human GPR-9-6) is administered to a subject toinhibit (i.e., reduce or prevent) inflammation. For example, antibodiesof the present invention, including mAb 3C3 can be used in the method.As a result, one or more inflammatory processes, such as leukocyteemigration, chemotaxis, exocytosis (e.g., of enzymes) or inflammatorymediator release, is inhibited. For example, leukocytic infiltration ofinflammatory sites (e.g., in a inflamed mucus membrane (e.g., colon,small intestine)) can be inhibited according to the present method. Inanother embodiment, an effective amount of an agent which inhibits oneor more functions of a mammalian GPR-9-6 protein (e.g., a human GPR-9-6)is administered to a subject to inhibit (i.e., reduce or prevent)GPR-9-6-mediated homing of leukocytes.

In another embodiment, an agent (e.g., receptor agonist) which promotes,one or more functions of a mammalian GPR-9-6 protein (e.g., a humanGPR-9-6) is administered to induce (trigger or enhance) the recruitmentof cells to a desired site or to induce an inflammatory response, suchas leukocyte emigration, chemotaxis, exocytosis (e.g., of enzymes) orinflammatory mediator release, resulting in the beneficial stimulationof inflammatory processes. For example, T cells can be recruited tocombat viral, bacterial or fungal infections.

Thus, the invention relates to a method of treating a subject having aninflammatory disease, comprising administering an effective amount of anantagonist of GPR-9-6 function. In a particular embodiment, the subjecthas an inflammatory bowel disease, such as Crohn's disease or colitis.

The invention also relates to a method of inhibiting GPR-9-6-mediatedhoming of leukocytes in a subject, comprising administering an effectiveamount of an antagonist of GPR-9-6 function, for example, the homing ofleukocytes to mucosal sites can be inhibited.

In a another embodiment, the invention relates to a method of promotingGPR-9-6 mediated homing of leukocytes in a subject, comprisingadministering an effective amount of a promoter (e.g., agonist) ofGPR-9-6 function.

The term “subject” is defined herein to include animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or otherbovine, ovine, equine, canine, feline, rodent or murine species.Diseases and conditions associated with inflammation and/or infectioncan be treated using the methods described herein. In a preferredembodiment, the disease or condition is one in which the actions oflymphocytes, particularly lymphocytes which home to mucosal tissues, areto be inhibited or promoted for therapeutic (including prophylactic)purposes. In a particularly preferred embodiment, the inflammatorydisease or condition is a T cell-mediated disease or condition.

Examples of inflammatory diseases associated with mucosal tissues whichcan be treated according to the present method include mastitis (mammarygland), vaginitis, cholecystitis, cholangitis or pericholangitis (bileduct and surrounding tissue of the liver), chronic bronchitis, chronicsinusitis, asthma, and graft versus host disease (e.g., in thegastrointestinal tract). As seen in Crohn's disease, inflammation oftenextends beyond the mucosal surface, accordingly chronic inflammatorydiseases of the lung which result in interstitial fibrosis, such asinterstitial lung diseases (ILD) (e.g., idiopathic pulmonary fibrosis,or ILD associated with rheumatoid arthritis, or other autoimmuneconditions), hypersensitivity pneumonitis, collagen diseases,sarcoidosis, and other idiopathic conditions can be amenable totreatment. Pancreatitis and insulin-dependent diabetes mellitus areother diseases which can be treated using the present method.

In a particularly preferred embodiment, diseases which can be treatedaccordingly include inflammatory bowel disease (IBD), such as ulcerativecolitis, Crohn's disease, ileitis, Celiac disease, nontropical Sprue,enteritis, enteropathy associated with seronegative arthropathies,microscopic or collagenous colitis, eosinophilic gastroenteritis, orpouchitis resulting after proctocolectomy, and ileoanal anastomosis.

Additional diseases or conditions, including chronic diseases, of humansor other species which can be treated with inhibitors of GPR-9-6function, include, but are not limited to:

inflammatory or allergic diseases and conditions, including systemicanaphylaxis or hypersensitivity responses, drug allergies (e.g., topenicillin, cephalosporins), insect sting allergies; psoriasis andinflammatory dermatoses such as dermatitis, eczema, atopic dermatitis,allergic contact dermatitis, urticaria; vasculitis (e.g., necrotizing,cutaneous, and hypersensitivity vasculitis); spondyloarthropathies;scleroderma; respiratory allergic diseases such as asthma, allergicrhinitis;

autoimmune diseases, such as arthritis (e.g., rheumatoid arthritis,psoriatic arthritis), multiple sclerosis, systemic lupus erythematosus,myasthenia gravis, juvenile onset diabetes, glomerulonephritis and othernephritides, autoimmune thyroiditis, Behcet's disease;

graft rejection (e.g., in transplantation), including allograftrejection or graft-versus-host disease;

other diseases or conditions in which undesirable inflammatory responsesare to be inhibited can be treated, including, but not limited to,atherosclerosis, myositis (including polymyositis, dermatomyositis).

Diseases or conditions of humans or other species which can be treatedwith promoters (e.g., an agonist) of GPR-9-6 function, include, but arenot limited to:

infectious diseases, such as bacterial infections and tuberculoidleprosy, and especially viral infections;

immunosuppression, such as that in individuals with immunodeficiencysyndromes such as AIDS, individuals undergoing radiation therapy,chemotherapy, or other therapy which causes immunosuppression;immunosuppression due congenital deficiency in receptor function orother causes.

Modes of Administration

According to the method, one or more agents can be administered to thesubject by an appropriate route, either alone or in combination withanother drug. An effective amount of an agent (e.g., a molecule whichinhibits ligand binding, an anti-GPR-9-6 antibody or antigen-bindingfragment thereof) is administered. An effective amount is an amountsufficient to achieve the desired therapeutic or prophylactic effect,under the conditions of administration, such as an amount sufficient forinhibition or promotion of GPR-9-6 receptor function, and thereby,inhibition or promotion, respectively, of a GPR-9-6-mediated process(e.g., an inflammatory response). The agents can be administered in asingle dose or multiple doses. The dosage can be determined by methodsknown in the art and is dependent, for example, upon the particularagent chosen, the subject's age, sensitivity and tolerance to drugs, andoverall well-being. Suitable dosages for antibodies can be from about0.01 mg/kg to about 100 mg/kg body weight per treatment.

A variety of routes of administration are possible including, forexample, oral, dietary, topical, transdermal, rectal, parenteral (e.g.,intravenous, intraarterial, intramuscular, subcutaneous injection,intradermal injection), and inhalation (e.g., intrabronchial, intranasalor oral inhalation, intranasal drops) routes of administration,depending on the agent and disease or condition to be treated.Administration can be local or systemic as indicated. The preferred modeof administration can vary depending upon the particular agent (e.g.,GPR-9-6 antagonist) chosen, and the particular condition (e.g., disease)being treated, however, oral or parenteral administration is generallypreferred.

The agent can be administered as a neutral compound or as a salt. Saltsof compounds containing an amine or other basic group can be obtained,for example, by reacting with a suitable organic or inorganic acid, suchas hydrogen chloride, hydrogen bromide, acetic acid, perchloric acid andthe like. Compounds with a quaternary ammonium group also contain acounteranion such as chloride, bromide, iodide, acetate, perchlorate andthe like. Salts of compounds containing a carboxylic acid or otheracidic functional group can be prepared by reacting with a suitablebase, for example, a hydroxide base. Salts of acidic functional groupscontain a countercation such as sodium, potassium and the like.

The agent can be administered to the individual as part of apharmaceutical composition for modulation of GPR-9-6 function comprisingan inhibitor or promotor of GPR-9-6 function and a pharmaceuticallyacceptable carrier. Formulation will vary according to the route ofadministration selected (e.g., solution, emulsion, capsule). Suitablepharmaceutical carriers can contain inert ingredients which do notinteract with the promoter (agonist) or inhibitor (antagonist) ofGPR-9-6 function. Standard pharmaceutical formulation techniques can beemployed, such as those described in Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. Suitable pharmaceuticalcarriers for parenteral administration include, for example, sterilewater, physiological saline, bacteriostatic saline (saline containingabout 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank'ssolution, Ringer's-lactate and the like. Methods for encapsulatingcompositions (such as in a coating of hard gelatin or cyclodextran) areknown in the art (Baker, et al., “Controlled Release of BiologicalActive Agents”, John Wiley and Sons, 1986). For inhalation, the agentcan be solubilized and loaded into a suitable dispenser foradministration (e.g., an atomizer, nebulizer or pressurized aerosoldispenser).

Furthermore, where the agent is a protein or peptide, the agent can beadministered via in vivo expression of the recombinant protein. In vivoexpression can be accomplished via somatic cell expression according tosuitable methods (see, e.g. U.S. Pat. No. 5,399,346). In thisembodiment, a nucleic acid encoding the protein can be incorporated intoa retroviral, adenoviral or other suitable vector (preferably, areplication deficient infectious vector) for delivery, or can beintroduced into a transfected or transformed host cell capable ofexpressing the protein for delivery. In the latter embodiment, the cellscan be implanted (alone or in a barrier device), injected or otherwiseintroduced in an amount effective to express the protein in atherapeutically effective amount.

The present invention will now be illustrated by the following Examples,which are not intended to be limiting in any way.

Exemplification

Purification of Cell Populations

Human peripheral blood was collected in 10% (v/v) 0.1 M EDTA, layeredonto 1-Step Polymorphs gradient (1.113±0.01 g/ml, Accurate Chemical Co.,Westbury, N.Y.) and centrifuged at 400×g for 30 minutes at roomtemperature. Neutrophil and mononuclear cell layers were collected,re-suspended in Dulbecco's phosphate buffered saline (DPBS) withoutcalcium and magnesium (Life Technologies, Grand Island, N.Y.) andcentrifuged for 15 minutes at 750×g. Red blood cells were lysed in theneutrophil fraction by re-suspending the pellet in E-Lyse (5 ml/10⁷cells)(Cardinal Associates, Santa Fe, N.M.) for 5 minutes on ice. Bothcell fractions were washed 2 times with ice cold DPBS. The mononuclearcells were allowed to adhere to protein coated plastic for 2-3 hours andthen non-adherent cells were gently washed off the plate. After afurther 12 hours the non-adherent dendritic cells were washed off theplate and depleted of B lymphocytes and T lymphocytes with anti-CD19 andanti-CD2 coated magnetic beads (Dynabeads; Dynal, Oslo, Norway) (5 beadsper cell). The remaining cells were cultured in 50 ng/ml granulocytemacrophage colony stimulating factor (GMCSF, R and D Systems,Minneapolis, Minn.) and 40 ng/ml IL-4 (R and D Systems) Dulbecco'smodified Eagle's medium (DMEM, Gibco BRL, Grand Island, N.Y.) 10% fetalcalf serum (FCS, HyClone, Logan, Utah) plus additives: penicillin 50U/ml, streptomycin 50 μg/ml, L-glutamine 2 mM, HEPES 10 mM, MEM sodiumpyruvate 10 mM, MEM nonessential amino acids 0.1 mM and2-mercaptoethanol 5.5×10⁻⁵M (all from Gibco BRL, Grand Island, N.Y.) for7 days (Sallusto, F. and Lanzabecchia, A., J. Exp. Med., 179:1109-1118(1994)) to generate immature dendritic cells (IMDC) and in some cases 24hours farther culture in 10 ng/ml LPS was used to mature the dendriticcells. CD4⁺, CD8⁺, CD14⁺, CD56⁺ and CD19⁺ populations were purified frommononuclear cells with the relevant Miltenyi Beads (Millenyi Biotek,Bergisch Gladbach, Germany) using 20 μl of beads for 10⁷ mononuclearcells in PBS/1% BSA/5 mM EDTA at 5×10⁷ cells/ml for 30 minutes at 4° C.They were then spun down, re-suspended in PBS/1% BSA/5 mM EDTA and 5×10⁷cells/ml and passed over a VS column (Miltenyi Biotech, Auburn, Calif.95603) in a magnetic field to remove non-tagged cells. Cells wereremoved by forcing 20 ml of PBS/1% BSA/5 mM EDTA over the VS column,outside the magnetic field.

Antibodies and Reagents

Labeled antibodies which bind to: CD4, CD8, CD14, CD19, CD49d, CD56,CD62L, CLA, CD45RA, CD45RO, CXCR5, CD80 and CD86 were obtained fromPharmingen (San Diego, Calif.) and used for immunofluorescence studies,while anti-αE and Anti-CD83 were obtained from Beckman Coulter(Fullerton, Calif.). OKT3, a anti-human CD3 mAb, was obtained fromAmerican Type Culture Collection (ATCC, Manassas, Va.) and anti-humanCD28 mAb was obtained from Becton Dickinson (Mountain View, Calif.).Some of the anti-chemokine receptor mAbs were produced at LeukoSite,Inc. (Cambridge, Mass.) and have the clone names anti-CCR3 (7B11),anti-CCR4 (2B10), anti-CCR6 (11A9) and anti-CXCR3 (1C6). Severalanti-chemokine receptor mAbs used in FACS analysis were obtained fromcommercial sources. Anti-CCR2, anti-CCR6 and anti-CXCR5 mAbs used forimmunofluorescence studies were obtained from R and D Systems(Minneapolis, Minn.), while anti-CCR5 and anti-CXCR4 were obtained fromPharmingen (San Diego, Calif.). Recombinant human chemokines wereobtained from Peprotech (Rocky Hill, N.J.) and R&D Systems (Minneapolis,Minn.) and in some cases synthesized using solid phase methods that wereoptimized and adapted to a fully automated peptide synthesizer (model430A; Applied Biosystems, Foster City, Calif.) as described(Clark-Lewis, I., et al., Biochemistry, 30:3128-3135 (1991)). The humanendothelial cell line ECV304 was purchased from ATCC. All cytokines wereobtained from R&D Systems (Minneapolis, Minn.).

Generation of Anti-GPR-9-6 mAbs

A peptide consisting of the NH₂ terminus of GPR-9-6 was generated havingthe sequence MADDYGSESTSSMEDYVNFNFTDFYC (SEQ ID NO:3). BALB/C mice wereimmunized i.p. with 10 μg of GPR-9-6 peptide/KLH conjugate prepared inFreunds Complete Adjutant (FCA, Sigma, St. Louis, Mo.) at day 1, 10 μgof GPR-9-6 peptide/KLH conjugate prepared in Incomplete Freunds Adjutant(IFA, Sigma, St. Louis, Mo.) at day 20, and 10 μg of GPR-9-6 peptide/KLHconjugate prepared in PBS at day 40. At day 60, the mice were boostedwith 10 μg of GPR-9-6 peptide/KLH in PBS, and after 4 days, the spleenswere removed and fused to SP2/0 myeloma cells (ATCC) (Coligan et al.,Current Protocols in Immunology 2.5.1 (1992)). Fusions were screened byELISA, using plates coated with GPR-9-6 peptide. Hybridomas producinganti-GPR-9-6 mAbs were checked for reactivity with GPR-9-6 transfectantsand subcloned for further characterization. Murine hybridoma 3C3, alsoreferred to as hybridoma LS129-3C3-E3-1, can be cultivated at 37° C. inan 5% CO2 atmosphere in DMEM supplemented with FCS (10%), IL-6 (100ng/ml), penicillin (50 U/ml), streptomycin (50 μg/ml), L-glutamine (2mM), HEPES (10 mM), MEM sodium pyruvate (10 mM), MEM nonessential aminoacids (0.1 mM) and 2-mercaptoethanol (5.5×10⁻⁵M).

Preparation of Chronically Activated T_(H)1 and T_(H)2 Lymphocytes

As previously described (Murphy, E., et al., J. Exp. Med., 183:901-913(1997)), six-well Falcon plates were coated overnight with 10 μg/mlanti-CD28 and 2 μg/ml OKT3, and then washed twice with PBS. Umbilicalcord blood CD4+ lymphocytes (Poietic Systems, German Town, Md.) werecultured at 10⁵-10⁶ cells/ml in DMEM with 10% FCS and IL-2 (4 ng/ml).IL-12 (5 ng/ml) and anti-IL-4 (1 μg/ml) were used to direct to T_(H)1,while IL-4 (5 ng/ml) and anti-IFN gamma (1 μg/ml) were used to direct toT_(H)2. After 4-5 days, the activated T_(H)1 and T_(H)2 lymphocytes werewashed once in DMEM and cultured for 4-7 days in DMEM with 10% FCS andIL-2 (1 ng/ml). Following this, the activated T_(H)1 and T_(H)2lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 andcytokines as described above, but with the addition of anti-CD95L (1μg/ml) to prevent apoptosis. After 4-5 days the T_(H)1 and T_(H)2lymphocytes were washed and then cultured again with IL-2 for 4 days.Activated T_(H)1 and T_(H)2 lymphocytes were maintained in this way fora maximum of three cycles.

ECV304 Transmigration and Chemotaxis Assays

3 micrometer pore diameter Transwell tissue culture inserts were eitherused uncoated or coated with 2% gelatin for 2 hours. Then 0.45 ml ofDMEM with 5% FCS was placed in the lower wells of the chambers and 2×10⁵EVC304 cells were added to each gelatin coated insert in 0.2 ml of DMEM5% FCS. After two days, the wells and inserts were washed twice withRPMI-1640 (Gibco BRL, Grand Island, N.Y.) containing 0.5% HSA (humanserum albumin), 10 mM HEPES and then chemokine was added to the lowerwell. The cells under study were washed once in RPMI and re-suspended at4×10⁶ cells/ml for T_(H)1/T_(H)2 lymphocytes, cell lines andtransfectants, or at 10⁷ cells/ml for resting CD4 lymphocytes in RPMI0.5% HSA and 10 mM HEPES. An aliquot of 200 μl of cell suspension (inputof 8×10⁵ cells and 2×10⁶ cells, respectively) was added to each insert.After 2 to 4 hours the inserts were removed and the number of cellswhich had migrated through the ECV304 monolayer to the lower wellcounted for 30 seconds on a Becton Dickinson FACScan with the gates setto acquire the cells of interest. Using this technique, 100% migrationwould be 25,000 cells for T_(H)1/T_(H)2 cells and 75,000 cells forresting CD4 lymphocytes, where this number represents the cells in thelower well counted on the FACScan over 1 minute. To study the phenotypeof migrating cells, identical experiments with CD4 lymphocytes wereperformed with 6 well plates using 24 mm diameter inserts. Chemotaxisassays were identical to ECV304 migration assays but Fibronectin coatedinserts (10 μg/ml) were used. In all cases, the data points were theresult of duplicate wells, with the mean value shown and the error barsrepresenting the sample standard deviation.

Ca²⁺ Mobilization (Ca²⁺ Flux) Assay

10⁷ cells/ml in DPBS were labeled for 30 minutes with Fura 2 dye(Molecular Probes, Eugene, Oreg.) at 2 mM, washed three times in DPBSand resuspended at 10⁶ cells/ml in DPBS containing 1 mM CaCl₂, 0.5 mMMgCl₂, 10 mM HEPES, and 5.5 mM glucose. The cells were then analyzed ona fluorimeter (Hitachi model F2000 fluorescence spectrophotometer,excitation 340 nm, emission 510 nm) using 10% NP-40 and 10 mM EDTA toestablish the max and min Ca²⁺ mobilizations.

Recombinant DNA Methods

Plasmid DNA was isolated using QIAGEN-tips as recommended by themanufacturer (QIAGEN Inc., Chatsworth, Calif.). DNA ligations,restriction endonuclease digestions, and gel electrophoresis wereperformed as described previously (Sambrook, J., et al., MolecularCloning: A Laboratory Manual 2nd ed., Cold Spring Harbor LaboratoryPress, (Cold Spring Harbor, N.Y.) (1989)). DNA purification throughagarose gel extraction was performed using the QIAEXII Gel ExtractionKit as recommended by the manufacturer (QIAGEN Inc., Chatsworth,Calif.). Plasmid DNA was introduced into E. coli by chemicaltransformation (GIBCO, Inc.). Enzymes were purchased from New EnglandBiolabs, Inc. (Beverly, Mass.), GIBCO Bethesda Research Laboratories,Inc. (Gaitherburg, Md.), or from Boehringer Mannheim, Inc. (Germany).RNA was isolated from frozen tissues or cells using either the standardguanidinium isothiocyanate method (Sambrook, J., et al., MolecularCloning: A Laboratory Manual 2nd ed., Cold Spring Harbor LaboratoryPress, (Cold Spring Harbor, N.Y.) (1989)) or the RNeasy kit asrecommended (QIAGEN Inc., Chatsworth, Calif.). DNA sequencing wasperformed by Sequi-Net (Colorado State University) using the FS DyeDeoxyTerminator cycle sequencing kit and a model 377 DNA sequencer (PerkinElmer Applied Biosystems, Foster City, Calif.). Sequences were analyzedusing SeqMan (DNASTAR, Inc., Madison, Wis.).

PCR

Primers were designed for use in the PCR to amplify the complete codingregion of GPR-9-6 based on the nucleotide sequence deposited in GenBank(U45982)(SEQ ID NO:1) which is incorporated herein by reference. BamHIand XbaI sites were incorporated into primer pair BAZ2015′.TCGAAGGGATCCCTAACATGGCTGATGACTATGGC.3′ (SEQ ID NO:4) and BAZ2025′.AAGAAGTCTAGAACCCCTCAGAGGGAGAGTGCTCC.3′ (SEQ ID NO:5) for directionalcloning (bold: coding sequence, italic: enzyme site). 5 μg of totalhuman genomic DNA (Clontech, Palo Alto, Calif.) was used as the templatein the Pfu PCR cycles, with 60 mM Tris-HCL, pH 9.5, 1.5 mM MgCl₂, 100pmol primers, 200 μM dNTP, and 5 units PfuI polymerase (Invitrogen,Carlsbad, Calif.) in a 100 μl volume. The cycle parameters were aninitial melt 95° C., 2 minutes, then 35 cycles: 95° C., 30 s; 55° C., 30s; 72° C., 2 minutes 15 s, followed by a final extension 72° C., 7minutes in a DNA thermal cycler (Perkin-Elmer Corp., Norwalk, Conn.).

Primers were designed to amplify the complete coding region of TECKbased on the published nucleotide sequence (accession U86358), which isincorporated herein by reference. HindIII and XbaI sites wereincorporated into primer pair BAZ2035′.TCGAAGAAGCTTATGAACCTGTGGCTCCTG.3′ (SEQ ID NO:6) and BAZ2045′.AAGAAGTCTAGATCACAGTCCTGAATTAGC.3′ (SEQ ID NO:7) for directionalcloning (bold: coding sequence, italic: enzyme site). 5 μg of humanthymus RNA was reverse transcribed with oligo dT in a 20 μl volume. ThecDNA was mixed with 200 μM dNTP, 100 pmol primers, 60 mM Tris-HCl, pH9.5, 1.5 mM MgCl₂, and 10 units AmpliTaq polymerase (Perkin-Elmer RocheMolecular Systems, Branchburg, N.J.) in a 50 μl volume. The cycleparameters were an initial melt 95° C., 2 minutes, then 35 cycles: 95°C., 30 s; 55° C., 30 s; 72° C., 1 minute, followed by a final extension72° C., 7 minutes The human thymus was obtained from Children's Hospital(Boston, Mass.).

Semi-quantitative PCR amplification of TECK using primers BAZ203 (SEQ IDNO:6) and BAZ204 (SEQ ID NO:7), and of GPR-9-6 using primers BAZ201 (SEQID NO:4) and BAZ202 (SEQ ID NO:5), was performed using equal amounts ofcDNA (500 ng) template from thymus, small intestine, colon, brain, lymphnode, and spleen, as well as 500 ng genomic DNA (ClonTech, Palo Alto,Calif.). The same conditions and PCR profile were used as the AmpliTaqPCR cycle described above, except that 30 cycles were performed.Amplification with glyceraldehyde-3-phosphate dehydrogenase (G3PDH)primers (ClonTech, Palo Alto, Calif., catalog number 5840-1) was used todemonstrate equivalency of template.

After agarose gel electrophoresis, the PCR products were visualized inthe presence of ethidium bromide with a UV light source. DNA fragmentsof predicted size (˜450 bp for TECK and 1 kb for GPR-9-6) were isolatedand cloned into pBluescript II KS+ (Stratagene, Inc., La Jolla, Calif.)and pcDNA3 (Stratagene, Inc.), respectively, for sequence analysis andfurther manipulation.

Expression Vector Construction and Generation of a GPR-9-6-ExpressingStable Cell Line

The coding region of GPR-9-6 was amplified by PCR and directionallycloned into the BamHI/XbaI sites of pcDNA3 (Invitrogen, San Diego,Calif.). Transfectants were then generated in the murine pre-B lymphomacell line L1.2, maintained in RPMI-1640 supplemented with 10% fetal calfserum (HyClone, Logan, Utah), 2 mM L-glutamine, 50 units/ml Pen/Strep,0.55 mM β-mercaptoethanol, 10 mM HEPES, and 1 mM sodium pyruvate (GibcoBRL). 20 μg of linearized GPR-9-6 in pcDNA3 was used to transfect thecell line as follows. L1.2 cells were washed twice in PBS andre-suspended in 0.8 ml of the same. The plasmid DNA was mixed with thecells and incubated for 10 minutes at room temperature, transferred to a0.4-cm electroporation cuvette, and a single pulse was then applied at250 V, 960 μF. The electroporation was followed by a 10-min incubationat room temperature. G418 (Geneticin, Gibco BRL) was added to a finalconcentration of 0.8 mg/ml 48 hours after transfection and the cellswere grown in bulk culture under drug selection 2-3 weeks. Thetransfectants were then stained by mAbs with reactivity against theGPR-9-6 peptide (see below) and analyzed by FACScan (Becton Dickinson &Co., Mountain View, Calif.) to confirm surface expression of GPR-9-6 andcloned by limiting dilution. Transfected cells were treated with 5 mMn-butyric acid for 24 hours before experimentation (Palmero, D. P., etal., J. Biotech., 19:35-47 (1991)).

Northern Blot Analysis

Northern blots were either purchased from ClonTech or prepared asfollows. Total RNA was separated by electrophoresis on 1.2% formaldehydeagarose gels and transferred to a nylon membranes (Hybond-N+; AmershamCorp., Arlington Heights, Ill.) by the capillary method as describedpreviously (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual2nd ed., Cold Spring Harbor Laboratory Press, (Cold Spring Harbor, N.Y.)(1989)) and crosslinked using a Stratalinker (Stratagene, Inc.).Hybridizations with radio-labeled probes was with ExpressHyb Solution(Clonetech) using the manufacture's suggested protocol. Length ofautoradiography exposure is described in appropriate figure legends.Full length gel purified TECK and GPR-9-6 DNA fragments were used inhybridizations.

Results

A mAb Raised to GPR-9-6; mAb 3C3 Selectively Reacts with GPR-9-6Transfectants

Due to its close phylogenetic association with other known leukocytechemokine receptors (FIG. 1), we cloned GPR-9-6 by PCR using primersdesigned from the deposited GenBank sequence. GPR-9-6/L1.2 transfectantswere prepared and stained with mAbs raised against GPR-9-6 in fusions inwhich mice had been immunized with the first 26 amino acids of the NH₂terminus of GPR-9-6 (SEQ ID NO:3) coupled to KLH. The mAb, designatedmAb 3C3, reacted with GPR-9-6/L1.2 transfectants but not with parentalL1.2 cells. mAb 3C3 was found to have an IgG_(2b) isotype. Incross-reactivity studies, mAb 3C3 did not cross-react with CCR1, CCR2,CCR3, CCR4, CCR5, CCR6, CCR7 or CXCR1, CXCR2, CXCR3 and CXCR4transfectants. The data for CCR6 are shown here, as it is one of themore closely related chemokine receptors to GPR-9-6 (FIGS. 2A-2B). Also,the NH₂ terminal peptide of GPR-9-6 (SEQ ID NO:3) was found tocompletely block the binding of mAb 3C3 to GPR-9-6 transfectants (datanot shown), further validating the specificity of this mAb.

GPR-9-6 is Expressed on All B Lymphocytes, Subsets of CD4 Lymphocytesand a Minor Subset of CD8 Lymphocytes in Peripheral Blood, as Well as onThymocytes

In initial two color studies of peripheral blood, GPR-9-6 was found tobe expressed on a small subset (2-4%) of CD4 lymphocytes as well as on avery small subset of CD8 lymphocytes, while B lymphocytes expressed lowand heterogeneous levels of GPR-9-6 (FIGS. 3A-3C). Monocytes, basophils,eosinophils, neutrophils and NK cells did not express GPR-9-6 under theconditions used (FIGS. 3D-3I). GPR-9-6 was expressed on a large subsetof thymocytes expressing all levels of TcR, although a small subset ofTcR^(high)GPR-9-6^(−ve) thymocytes was evident. In three-colorexperiments, GPR-9-6 was found on the majority of CD4, CD8 andCD4^(+ve)CD8^(+ve) thymocytes and on approximately 50% of immatureCD4^(−ve)CD8^(−ve) thymocytes (data not shown). No expression of GPR-9-6was seen on either immature or mature dendritic cells (FIG. 4D).However, as expected, immature dendritic cells expressed CCR5, which wasdown-regulated on LPS activation, while CD83 and CD86 were up-regulated(FIGS. 4A-4C). In examining a large panel of cell lines GPR-9-6 wasfound on several T cell lines (Table 1). Umbilical CD4+ lymphocytes didnot express GPR-9-6 (FIG. 4E) and chronic activation of these cells inthe presence of IL-12 or IL-4 to generate T_(H)1 or T_(H)2 lymphocytesfailed to induce the expression of GPR-9-6 (FIG. 4H). However, asexpected, CXCR3 were clearly up-regulated on T_(H)1 lymphocytes (FIG.4F), while α4β7, an integrin utilized in lymphocyte trafficking tomucosal sites, was up-regulated on both T_(H)1 and T_(H)2 lymphocytes(FIG. 4G).

Expression of GPR-9-6 on CD4 lymphocytes and B lymphocytes was measuredover time, and was found to be relatively constant (FIG. 5A). However,activation of T lymphocytes with anti-CD3 mAb resulted in transientdown-regulation of GPR-9-6 over 2 days, with expression recovering after10 days of culture in IL-2 (FIG. 5B). Chemokine receptors CCR6 and CCR5showed similar changes in expression upon T lymphocyte activation (FIG.5C).

The CD4 Lymphocyte Subset That Express GPR-9-6 are Predominantly ofMemory Phenotype and Express High Levels of Mucosal Lymphoid HomingReceptor α4β7 But Not Skin Homing Receptor CLA

The small subset of CD4 lymphocytes that express GPR-9-6 were examinedin more detail by three-color staining (FIGS. 6A-6F). The CD4lymphocytes that express GPR-9-6 were mainly of memory phenotype, andthose cells that expressed the highest levels of GPR-9-6 were all ofmemory phenotype. Interestingly, memory CLA^(+ve) CD4 lymphocytes, whichtraffic to skin, did not express GPR-9-6. In contrast, a subset ofmemory α4β7^(high) CD4 lymphocytes, which traffic to mucosal sites,clearly expressed GPR-9-6. The subset of memory CD4 lymphocytes definedby expression of αEβ7 were also clearly subdivided into GPR-9-6 positiveand negative subsets. GPR-9-6^(high) CD4 lymphocytes did not expressCD62L, a homing receptor which is involved in trafficking to peripherallymph nodes, while a small subset of GPR-9-6^(dull)CD62L^(+ve)lymphocytes was evident.

GPR-9-6^(+ve) CD4 lymphocytes were also examined for co-expression ofother chemokine receptors known to be expressed on CD4 lymphocytes(FIGS. 7A-7F). While GPR-9-6 was clearly found on both positive andnegative subsets of CCR5, CCR6, CXCR3 and CXCR5, CD4 lymphocyteexpression of CCR2 and GPR-9-6 was mutually exclusive.

GPR-9-6 Chemokine Receptor Specifically Binds to TECK

Out of all the published chemokines tested, only TECK proved able toinduce chemotaxis of GPR-9-6/L1.2 transfectants (FIG. 8A). MCP-1-4,MIP-1α, MIP-1β, eotaxin-1, eotaxin-2, RANTES, I-309, TARC, MDC, MIP4,SLC, HCC1, fractalkine, lymphotactin, MIG, IP-10, ITAC, ADEC, IL-S,gro-α, gro-β, gro-γ, leukotactin, SDF-1α, SDF-1β, MIP3 and MIP4 allproved unable to induce chemotaxis of the GPR-9-6/L1.2 transfectants.TECK induced chemotaxis of L1.2/GPR-9-6 transfectants was inhibited bythe mAb 3C3, but not by an anti-CCR3 mAb 7B11 (FIG. 8B). TECK did notact on any of the other transfectants tested (CCR1, CCR2, CCR 4, CCR5,CCR6, CCR7 and CXCR1, CXCR2, CXCR3, CXCR4, data not shown).Interestingly, TECK was also found to act on the T cell lines MOLT-4(FIG. 8D) and MOLT-13 (FIG. 8F), which express GPR-9-6 (Table 1). TECKwas not chemotactic for other cell lines, such as SKW3 (FIG. 8E), whichdo not express GPR-9-6. Using the T cell line MOLT-4, TECK inducedchemotaxis was shown to be blocked by pertussis toxin (FIG. 8C).Additionally, the anti-GPR-9-6 mAb 3C3 blocked the chemotaxis of theMOLT-13 cells to TECK, but had no effect on SDF1α induced chemotaxis ofthese cells (FIG. 8F). In calcium mobilization experiments, TECK wasalso found to induce Ca²⁺ flux in GPR-9-6^(+ve) cell lines such asMOLT-4 (FIGS. 9A-9C), while chemokines such as MDC for which these cellsdo not express the relevant receptor had no effect.

TABLE 1 GPR-9-6 Expression by Cell Lines CELL GPR-9-6 CXCR4 MOLT-4 + +MOLT-13 + + CEM − + PEER − + HUT78 − + PMI − + SKW.3 − + JURKAT − +RAMOS − + RAJI − + JY − + THP-1 − − U937 − + KG1 − − HL-60 − +/− K562 −− EOL-1 − + KU812 − −

Leukocyte subsets were also tested (FIGS. 10A-10F) to determine if theychemotaxed to TECK. As observed in the mouse, neutrophils, monocytes,eosinophils, CD8 and NK cells did not chemotax to TECK, but did chemotaxto other chemokines. However, TECK was chemotactic for a minor subset ofCD4 lymphocytes. As murine TECK induces thymocyte chemotaxis, chemotaxisof human thymocytes to TECK and SDF1α, both of which mediate thymocytechemotaxis (data not shown) was examined. Anti-GPR-9-6 mAb 3C3 blockedthymocyte and CD4 lymphocyte chemotaxis to TECK. The anti-GPR-9-6 mAb3C3 had no effect on TARC-induced chemotaxis of CD4 lymphocytes,indicating that the effect is specific (FIGS. 11A-11C). These resultsindicate that GPR-9-6 is the major physiological receptor for TECK.

Tissue Distribution of TECK and GPR-9-6 Transcripts

Due to the expression of GPR-9-6 on mucosal homing lymphocytes, thedistribution of TECK and GPR-9-6 transcripts in lymphoid and mucosaltissue was examined (FIGS. 12A-12B). TECK was selectively expressed inthymus and small intestine (FIG. 12A), while GPR-9-6 was expressed athigh levels in thymus and weakly in spleen and peripheral bloodleukocytes (FIG. 12B). While GPR-9-6 transcripts were not detected byNorthern blot analysis in small intestine, GPR-9-6 message was detectedin small intestine, thymus, lymph node and spleen using the moresensitive technique of RT-PCR (FIG. 12C). Messages for both TECK andGPR-9-6 were not detected in brain or colon. In other Northern blots,TECK and GPR-9-6 were not detected in T_(H)1, T_(H)2, Tr1 (Groux, etal., Nature 389:737-742 (1997)) lymphocytes, LAK cells, monocytes, CD34derived dendritic cells, monocyte derived dendritic cells, astrocytes,human umbilical vein endothelial cells (HUVEC) and pulmonary veinendothelial cells (PUVEC) (data not shown). Finally, GPR-9-6 transcriptwas shown to be present only in cell lines which had previously beenshown to be GPR-9-6⁺ by staining with mAb 3C3, further validating thespecificity of the mAb (FIG. 12B).

Only α4β7^(high) CD4 and CD8 Lymphocytes Migrate to TECK

As GPR-9-6 is expressed mainly on memory α4β7^(high) CD4 lymphocytes,CD45RA^(−ve) memory CD4 and CD8 lymphocytes which expressed none,intermediate or high levels of α4β7 were isolated. Only α4β7^(high)memory CD8 lymphocytes and α4β7^(+ve) CLA^(−ve) memory CD4 lymphocyteschemotaxed to TECK (FIGS. 13A-13B).

Discussion

Several different adhesion molecules are involved in trafficking oflymphocyte subsets to distinct physiologic location, such as peripherallymph node (Gallatin, W. M., et al., Nature, 304:30-34 (1983)), Peyer'sPatches (Hamman, A., et al., J. Immunol., 152:3282-3292 (1994); Andrew,D. P., et al., Eur. J. Immunol., 26:897-905 (1996)) and inflammatorysites (Frenette, P. S., et al., Cell, 84:563-574 (1996); Tietz, W. Y.,et al., J. Immunol., 161(2):963-970 (1998); Picker, L. J., et al., J.Immunol., 145:3247-3255 (1990)). It is thought that specific chemokinereceptors expressed on these lymphocyte subsets may interact withchemokines expressed in the areas mediating leukocyte activation,arrest, and transendothelial migration. It is therefore possible thatCD4 subsets defined by the expression of certain adhesion molecules, mayalso express known, orphan or as yet undiscovered chemokine receptorsthat are important for trafficking of the lymphocytes into these sites.The work described herein relates to one such chemokine receptor thatmay be involved in the selective trafficking of memory CD4 and CD8lymphocyte subsets to mucosal sites.

GPR-9-6 was originally chosen as a potentially interesting orphanchemokine receptor due to its strong phylogenetic linkage with otherknown chemokine receptors including CCR6 and CCR7. In Northern blotanalysis, GPR-9-6 was found in thymus, indicative of some role in T celldevelopment. The weak expression in spleen and blood may reflect theexpression of GPR-9-6 on memory T lymphocytes and B lymphocytes. AsGPR-9-6 is expressed by the majority of thymocytes, and theseGPR-9-6^(+ve) thymocytes express all levels of TcR, GPR-9-6 isapparently expressed at all stages of T cell development. On exit fromthe thymus, GPR-9-6 must be down-regulated, as in the periphery only asmall subset of CD4 lymphocytes and an even smaller subset of CD8lymphocytes express GPR-9-6. In three-color experiments, GPR-9-6 isfound predominantly on memory CD4 lymphocytes. Of greater interest,while the CLA^(+ve) memory CD4 lymphocytes (Picker, L. J., et al., J.Immunol., 145:3247-3255 (1990)) do not express GPR-9-6, a subset of thememory α4β7^(high) CD4 lymphocytes (Andrew, D. P., et al., Eur. J.Immunol., 26:897-905 (1996)) express this chemokine receptor. This mayreflect a role for GPR-9-6 in the trafficking of lymphocytes to mucosalsites, or their effector action when there. While GPR-9-6 was clearlyexpressed on mucosal trafficking CD4 lymphocytes, GPR-9-6 transcriptswere not detected in small intestine by Northern blot analysis. This mayreflect the low numbers of the GPR-9-6^(+ve) CD4+ and/or CD8+lymphocytes in small intestine tissue compared to thymus, where themajority of the cells are actively dividing GPR-9-6^(+ve) thymocytes.However, using the more sensitive technique of RT-PCR, GPR-9-6transcripts were detected in small intestine but not in the brain.Interestingly, while GPR-9-6 and TECK transcripts are expressed in smallintestine, GPR-9-6 or TECK transcripts were not detected in the colon byeither Northern or RT-PCR analysis.

It is possible that factors present in the mucosal environment lead tothe induction of GPR-9-6 on T lymphocytes as well as TECK expression.Cytokines present in T_(H)1/T_(H)2 environments induce expression ofcertain chemokine receptors, such as CCR4 on T_(H)2 and CXCR3 on T_(H)1lymphocytes, as well as the production of the chemokines that bind thesereceptors (Bonecchi, R. G., et al., J. Exp. Med., 187:129-134 (1998);Sallusto, F. D., et al., J. Exp. Med., 187:875-883 (1998); Sallusto, F.,Science, 277:2005-2007 (1997); Andrew, D. P., et al., (1998); Zingoni,A., et al., J. Immunol., 161:547-555 (1998)). However, these conditionsdid not up-regulate GPR-9-6 expression on T lymphocytes. Also, attemptsto induce expression of GPR-9-6 on activated umbilical CD4 lymphocyteswith cytokines IL-1-18 or with TGF-β, previously shown to induce αE on Tlymphocytes (Kilshaw, P. J. and Murant, S. J., Eur. J. Immunol.,21:2591-2597 (1991)), failed to identify a cytokine that up-regulatesGPR-9-6 expression. Therefore, the mechanism by which GPR-9-6 expressionis controlled on CD4 lymphocytes is unclear. Upon activation via TcRcross-linking, expression of GPR-9-6 is down-regulated, as is theexpression of chemokine receptor CXCR4 (Bermejo, M., et al., J. Immunol.28:3192-3204 (1998)). As TcR cross-linking mimics antigen presentation,we conclude that on entering a lymph node and encountering APC'sexpressing antigenic peptide+MHC-II, that T lymphocytes willdown-regulate chemokine receptors such as GPR-9-6. This will hold Tlymphocytes in the lymph node, where T lymphocytes may mediate otherimmune functions such as B cell class switching through T:B cognateinteractions.

Out of all the chemokines tested only TECK (Vicari, A. P., et al.,Immunity, 7(2):291-301 (1997)) acted as a chemoattractant forGPR-9-6/L1.2 transfectants, with 150 nM resulting in optimal chemotaxis.This falls into the range of 1 nM-1 μM for which other leukocytechemokines are active. However, as we are using TECK that was generatedby peptide synthesis, we cannot be sure that either post-translationalmodifications or further cleavage of TECK by factors outside the cell invivo do not generate more active fragments, as is the case for CKB8(Macphee, C. H., et al., J. Immunol. 161:6273-6279 (1998)). TECK did notact as a chemoattractant for CCR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR9and CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5 L1.2 transfectants. However,some weak activity or TECK on CCR3/L1.2 transfectants which wasapproximately 20% of the chemotactic activity observed with eotaxin-1was detected. This activity was blocked by anti-CCR3 mAbs, though TECKdid not act as a chemoattractant for eosinophils. Therefore, TECK isprobably not a physiological chemokine for the CCR3 receptor. Thisresult is not unprecedented, as in previous studies MIP-1α has beenshown to act as a chemoattractant for CCR4/HEK293 transfectants (Power,C. A., et al., J. Biol. Chem., 270:19495-19500 (1995)), but notCCR4/L1.2 transfectants (Imai, T. M., et al., J. Bid. Chem.,272:15036-15042 (1997)). In further experiments, only the T cell linesthat express GPR-9-6 were found to chemotax to TECK, while among primarycells TECK was chemotactic for only a small subset of CD4 lymphocytes.Presumably, these cells represent the small subset of CD4 lymphocytesthat express GPR-9-6, as the chemotaxis was blocked by anti-GPR-9-6 mAb3C3. Additionally, only α4β7^(+ve) memory CD4 and CD8 lymphocyteschemotax to TECK, which would be the subset predicted to expressGPR-9-6. TECK was originally described as a chemokine produced by thymicdendritic cell, whose expression is restricted to thymus and smallintestine (Vicari, A. P., et al., Immunity, 7(2):291-301 (1997)). OurNorthern data confirms this observation and shows that the receptor forTECK, GPR-9-6, is also expressed at these sites. The expression of bothchemokine receptor GPR-9-6 and its ligand TECK in small intestine andthymus predict a role for GPR-9-6 and TECK in T cell development andmucosal immunology.

In summary, the orphan chemokine receptor GPR-9-6 was shown to beexpressed on the majority of thymocytes and on a subset of memory CD4lymphocytes that traffic to mucosal sites. The selective expression ofTECK and GPR-9-6 in thymus and small intestine imply a dual role forGPR-9-6, both in T cell development and the mucosal immune response.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 7 <210> SEQ ID NO 1 <211> LENGTH: 2577<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (58)...(1131) <400> SEQUENCE: 1aatattttcc ttgacctaat gccatcttgt gtccccttgc agagccctat tc#ctaac atg    60                    #                  #                   #         Met                    #                  #                   #          1gct gat gac tat ggc tct gaa tcc aca tct tc#c atg gaa gac tac gtt      108Ala Asp Asp Tyr Gly Ser Glu Ser Thr Ser Se #r Met Glu Asp Tyr Val             5      #              10     #              15aac ttc aac ttc act gac ttc tac tgt gag aa#a aac aat gtc agg cag      156Asn Phe Asn Phe Thr Asp Phe Tyr Cys Glu Ly #s Asn Asn Val Arg Gln         20          #         25          #         30ttt gcg agc cat ttc ctc cca ccc ttg tac tg#g ctc gtg ttc atc gtg      204Phe Ala Ser His Phe Leu Pro Pro Leu Tyr Tr #p Leu Val Phe Ile Val     35              #     40              #     45ggt gcc ttg ggc aac agt ctt gtt atc ctt gt#c tac tgg tac tgc aca      252Gly Ala Leu Gly Asn Ser Leu Val Ile Leu Va #l Tyr Trp Tyr Cys Thr 50                  # 55                  # 60                  # 65aga gtg aag acc atg acc gac atg ttc ctt tt#g aat ttg gca att gct      300Arg Val Lys Thr Met Thr Asp Met Phe Leu Le #u Asn Leu Ala Ile Ala                 70  #                 75  #                 80gac ctc ctc ttt ctt gtc act ctt ccc ttc tg#g gcc att gct gct gct      348Asp Leu Leu Phe Leu Val Thr Leu Pro Phe Tr #p Ala Ile Ala Ala Ala             85      #             90      #             95gac cag tgg aag ttc cag acc ttc atg tgc aa#g gtg gtc aac agc atg      396Asp Gln Trp Lys Phe Gln Thr Phe Met Cys Ly #s Val Val Asn Ser Met        100           #       105           #       110tac aag atg aac ttc tac agc tgt gtg ttg ct#g atc atg tgc atc agc      444Tyr Lys Met Asn Phe Tyr Ser Cys Val Leu Le #u Ile Met Cys Ile Ser    115               #   120               #   125gtg gac agg tac att gcc att gcc cag gcc at#g aga gca cat act tgg      492Val Asp Arg Tyr Ile Ala Ile Ala Gln Ala Me #t Arg Ala His Thr Trp130                 1 #35                 1 #40                 1 #45agg gag aaa agg ctt ttg tac agc aaa atg gt#t tgc ttt acc atc tgg      540Arg Glu Lys Arg Leu Leu Tyr Ser Lys Met Va #l Cys Phe Thr Ile Trp                150   #               155   #               160gta ttg gca gct gct ctc tgc atc cca gaa at#c tta tac agc caa atc      588Val Leu Ala Ala Ala Leu Cys Ile Pro Glu Il #e Leu Tyr Ser Gln Ile            165       #           170       #           175aag gag gaa tcc ggc att gct atc tgc acc at#g gtt tac cct agc gat      636Lys Glu Glu Ser Gly Ile Ala Ile Cys Thr Me #t Val Tyr Pro Ser Asp        180           #       185           #       190gag agc acc aaa ctg aag tca gct gtc ttg ac#c ctg aag gtc att ctg      684Glu Ser Thr Lys Leu Lys Ser Ala Val Leu Th #r Leu Lys Val Ile Leu    195               #   200               #   205ggg ttc ttc ctt ccc ttc gtg gtc atg gct tg#c tgc tat acc atc atc      732Gly Phe Phe Leu Pro Phe Val Val Met Ala Cy #s Cys Tyr Thr Ile Ile210                 2 #15                 2 #20                 2 #25att cac acc ctg ata caa gcc aag aag tct tc#c aag cac aaa gcc cta      780Ile His Thr Leu Ile Gln Ala Lys Lys Ser Se #r Lys His Lys Ala Leu                230   #               235   #               240aaa gtg acc atc act gtc ctg acc gtc ttt gt#c ttg tct cag ttt ccc      828Lys Val Thr Ile Thr Val Leu Thr Val Phe Va #l Leu Ser Gln Phe Pro            245       #           250       #           255tac aac tgc att ttg ttg gtg cag acc att ga#c gcc tat gcc atg ttc      876Tyr Asn Cys Ile Leu Leu Val Gln Thr Ile As #p Ala Tyr Ala Met Phe        260           #       265           #       270atc tcc aac tgt gcc gtt tcc acc aac att ga#c atc tgc ttc cag gtc      924Ile Ser Asn Cys Ala Val Ser Thr Asn Ile As #p Ile Cys Phe Gln Val    275               #   280               #   285acc cag acc atc gcc ttc ttc cac agt tgc ct#g aac cct gtt ctc tat      972Thr Gln Thr Ile Ala Phe Phe His Ser Cys Le #u Asn Pro Val Leu Tyr290                 2 #95                 3 #00                 3 #05gtt ttt gtg ggt gag aga ttc cgc cgg gat ct#c gtg aaa acc ctg aag     1020Val Phe Val Gly Glu Arg Phe Arg Arg Asp Le #u Val Lys Thr Leu Lys                310   #               315   #               320aac ttg ggt tgc atc agc cag gcc cag tgg gt#t tca ttt aca agg aga     1068Asn Leu Gly Cys Ile Ser Gln Ala Gln Trp Va #l Ser Phe Thr Arg Arg            325       #           330       #           335gag gga agc ttg aag ctg tcg tct atg ttg ct#g gag aca acc tca gga     1116Glu Gly Ser Leu Lys Leu Ser Ser Met Leu Le #u Glu Thr Thr Ser Gly        340           #       345           #       350gca ctc tcc ctc tga ggggtcttct ctgaggtgca tggttcttt#t ggaagaaatg     1171 Ala Leu Ser Leu  *     355agaaatacat gaaacagttt ccccactgat gggaccagag agagtgaaag ag#aaaagaaa   1231actcagaaag ggatgaatct gaactatatg attacttgta gtcagaattt gc#caaagcaa   1291atatttcaaa atcaactgac tagtgcagga ggctgttgat tggctcttga ct#gtgatgcc   1351cgcaattctc aaaggaggac taaggaccgg cactgtggag caccctggct tt#gccactcg   1411ccggagcatc aatgccgctg cctctggagg agcccttgga ttttctccat gc#actgtgaa   1471cttctgtggc ttcagttctc atgctgcctc ttccaaaagg ggacacagaa gc#actggctg   1531ctgctacaga ccgcaaaagc agaaagtttc gtgaaaatgt ccatctttgg ga#aattttct   1591accctgctct tgagcctgat aacccatgcc aggtcttata gattcctgat ct#agaacctt   1651tccaggcaat ctcagaccta atttccttct gttctccttg ttctgttctg gg#ccagtgaa   1711ggtccttgtt ctgattttga aacgatctgc aggtcttgcc agtgaacccc tg#gacaactg   1771accacaccca caaggcatcc aaagtctgtt ggcttccaat ccatttctgt gt#cctgctgg   1831aggttttaac ctagacaagg attccgctta ttccttggta tggtgacagt gt#ctctccat   1891ggcctgagca gggagattat aacagctggg ttcgcaggag ccagccttgg cc#ctgttgta   1951ggcttgttct gttgagtggc acttgctttg ggtccaccgt ctgtctgctc cc#tagaaaat   2011gggctggttc ttttggccct cttctttctg aggcccactt tattctgagg aa#tacagtga   2071gcagatatgg gcagcagcca ggtagggcaa aggggtgaag cgcaggcctt gc#tggaaggc   2131tatttacttc catgcttctc cttttcttac tctatagtgg caacatttta aa#agctttta   2191acttagagat taggctgaaa aaaataagta atggaattca cctttgcatc tt#ttgtgtct   2251ttcttatcat gatttggcaa aatgcatcac ctttgaaaat atttcacata tt#ggaaaagt   2311gctttttaat gtgtatatga agcattaatt acttgtcact ttctttaccc tg#tctcaata   2371ttttaagtgt gtgcaattaa agatcaaata gatacattaa gagtgtgaag gc#tggtctga   2431aggtagtgag ctatctcaat cggattgttc acactcagtt acagattgaa ct#ccttgttc   2491tacttccctg cttctctcta ctgcaattga ctagtcttta aaaaaaagtg tg#aagagtaa   2551 gcaataggga taaggaaata agatct          #                   #            2577 <210> SEQ ID NO 2<211> LENGTH: 357 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 2 Met Ala Asp Asp Tyr Gly Ser Glu Ser Thr Se#r Ser Met Glu Asp Tyr  1               5   #                10  #                15 Val Asn Phe Asn Phe Thr Asp Phe Tyr Cys Gl#u Lys Asn Asn Val Arg             20       #            25      #            30 Gln Phe Ala Ser His Phe Leu Pro Pro Leu Ty#r Trp Leu Val Phe Ile         35           #        40          #        45 Val Gly Ala Leu Gly Asn Ser Leu Val Ile Le#u Val Tyr Trp Tyr Cys     50               #    55              #    60 Thr Arg Val Lys Thr Met Thr Asp Met Phe Le#u Leu Asn Leu Ala Ile 65                   #70                  #75                   #80 Ala Asp Leu Leu Phe Leu Val Thr Leu Pro Ph#e Trp Ala Ile Ala Ala                 85   #                90  #                95 Ala Asp Gln Trp Lys Phe Gln Thr Phe Met Cy#s Lys Val Val Asn Ser             100       #           105      #           110 Met Tyr Lys Met Asn Phe Tyr Ser Cys Val Le#u Leu Ile Met Cys Ile         115           #       120          #       125 Ser Val Asp Arg Tyr Ile Ala Ile Ala Gln Al#a Met Arg Ala His Thr     130               #   135              #   140 Trp Arg Glu Lys Arg Leu Leu Tyr Ser Lys Me#t Val Cys Phe Thr Ile 145                 1 #50                 1#55                 1 #60 Trp Val Leu Ala Ala Ala Leu Cys Ile Pro Gl#u Ile Leu Tyr Ser Gln                 165   #               170  #               175 Ile Lys Glu Glu Ser Gly Ile Ala Ile Cys Th#r Met Val Tyr Pro Ser             180       #           185      #           190 Asp Glu Ser Thr Lys Leu Lys Ser Ala Val Le#u Thr Leu Lys Val Ile         195           #       200          #       205 Leu Gly Phe Phe Leu Pro Phe Val Val Met Al#a Cys Cys Tyr Thr Ile     210               #   215              #   220 Ile Ile His Thr Leu Ile Gln Ala Lys Lys Se#r Ser Lys His Lys Ala 225                 2 #30                 2#35                 2 #40 Leu Lys Val Thr Ile Thr Val Leu Thr Val Ph#e Val Leu Ser Gln Phe                 245   #               250  #               255 Pro Tyr Asn Cys Ile Leu Leu Val Gln Thr Il#e Asp Ala Tyr Ala Met             260       #           265      #           270 Phe Ile Ser Asn Cys Ala Val Ser Thr Asn Il#e Asp Ile Cys Phe Gln         275           #       280          #       285 Val Thr Gln Thr Ile Ala Phe Phe His Ser Cy#s Leu Asn Pro Val Leu     290               #   295              #   300 Tyr Val Phe Val Gly Glu Arg Phe Arg Arg As#p Leu Val Lys Thr Leu 305                 3 #10                 3#15                 3 #20 Lys Asn Leu Gly Cys Ile Ser Gln Ala Gln Tr#p Val Ser Phe Thr Arg                 325   #               330  #               335 Arg Glu Gly Ser Leu Lys Leu Ser Ser Met Le#u Leu Glu Thr Thr Ser             340       #           345      #           350 Gly Ala Leu Ser Leu         355 <210> SEQ ID NO 3<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: NH2-Terminal Peptide of H#uman GPR-9-6 <400> SEQUENCE: 3Met Ala Asp Asp Tyr Gly Ser Glu Ser Thr Se #r Ser Met Glu Asp Tyr 1               5   #                10   #                15Val Asn Phe Asn Phe Thr Asp Phe Tyr Cys             20      #            25 <210> SEQ ID NO 4 <211> LENGTH: 35 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 4tcgaagggat ccctaacatg gctgatgact atggc        #                  #       35 <210> SEQ ID NO 5 <211> LENGTH: 35 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 5aagaagtcta gaacccctca gagggagagt gctcc        #                  #       35 <210> SEQ ID NO 6 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 6tcgaagaagc ttatgaacct gtggctcctg          #                  #           30 <210> SEQ ID NO 7 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 7aagaagtcta gatcacagtc ctgaattagc          #                  #           30

What is claimed is:
 1. A method of detecting or identifying an agentwhich binds a mammalian GPR-9-6 comprising combining: a) TECK, b) a testagent, and c) a composition comprising a protein, said proteincomprising GPR-9-6, under conditions suitable for binding of said TECKto said GPR-9-6, wherein said GPR-9-6 binds TECK and comprises an aminoacid sequence that is at least about 90% similar to the amino acidsequence of SEQ ID NO:2; and detecting or measuring the formation of acomplex between said TECK and said GPR-9-6, wherein a decrease in theformation of said complex relative to a suitable control indicates thatsaid test agent binds a mammalian GPR-9-6.
 2. The method of claim 1wherein said TECK is labeled with a label selected from the groupconsisting of a radioisotope, an epitope, an affinity label, an enzyme,a fluorescent group and a chemiluminescent group.
 3. The method of claim1 wherein said composition comprising a protein is a cell that expressessaid protein comprising GPR-9-6.
 4. The method of claim 3 wherein saidcell is a recombinant cell.
 5. The method of claim 3 wherein said cellis a cell line.
 6. The method of claim 5 wherein said cell line isselected from the group consisting of MOLT-4 and MOLT-13.
 7. The methodof claim 3 wherein the formation of a complex between said TECK and saidGPR-9-6 is detected or measured by monitoring chemotaxis or Ca²⁺ fluxinduced upon binding of said TECK to the protein comprising GPR-9-6expressed on said cell.
 8. The method of claim 1 wherein saidcomposition comprising a protein is a membrane preparation of a cellthat expresses said protein comprising GPR-9-6.
 9. The method of claim 1wherein said GPR-9-6 is a human GPR-9-6.
 10. The method of claim 1wherein said GPR-9-6 comprises the amino acid sequence of SEQ ID NO:2.11. The method of claim 1 wherein said test agent is an organiccompound.
 12. The method of claim 1 wherein said test agent is anantibody or antigen-binding fragment of an antibody.
 13. The method ofclaim 1 wherein said test agent is a peptide.
 14. The method of claim 1wherein said test agent is a nucleic acid.
 15. A method of detecting oridentifying an agent which binds a mammalian GPR-9-6 comprisingcombining: a) a composition comprising a protein, said proteincomprising GPR-9-6, wherein said GPR-9-6 binds TECK and comprises anamino acid sequence that is at least about 90% similar to the amino acidsequence of SEQ ID NO:2, b) a test agent, and c) an antibody orantigen-binding fragment thereof which binds SEQ ID NO:2 and inhibitsbinding of TECK to SEQ ID NO:2, under conditions suitable for binding ofsaid antibody or antigen-binding fragment to said GPR-9-6, and detectingor measuring the formation of a complex between said antibody orantigen-binding fragment and said GPR-9-6, wherein a decrease in theformation of said complex relative to a suitable control indicates thatsaid test agent binds to a mammalian GPR-9-6.
 16. The method of claim 15wherein said antibody or antigen-binding fragment is labeled with alabel selected from the group consisting of a radioisotope, an epitope,an affinity label, an enzyme, a fluorescent group and a chemiluminescentgroup.
 17. The method of claim 15 wherein the binding of said antibodyor antigen-binding fragment to said GPR-9-6 is inhibited by a peptidethat consists of the amino acid sequence of SEQ ID NO:3.
 18. The methodof claim 15 wherein said antibody or antigen-binding fragment has theepitopic specificity of mAb 3C3 (ATCC Accession No. HB-12653).
 19. Themethod of claim 15 wherein said composition comprising a protein is acell that expresses said protein comprising GPR-9-6.
 20. The method ofclaim 19 wherein said cell is a recombinant cell.
 21. The method ofclaim 19 wherein said cell is a cell line.
 22. The method of claim 21wherein said cell line is selected from the group consisting of MOLT-4and MOLT-13.
 23. The method of claim 19 wherein the formation of acomplex between said antibody or antigen-binding fragment and saidGPR-9-6 is detected or measured by monitoring chemotaxis or Ca²⁺ fluxinduced upon binding of said antibody or antigen-binding fragment to theprotein comprising GPR-9-6 expressed on said cell.
 24. The method ofclaim 15 wherein said composition comprising a protein is a membranepreparation of a cell that expresses said protein comprising GPR-9-6.25. The method of claim 15 wherein said GPR-9-6 is a human GPR-9-6. 26.The method of claim 15 wherein said GPR-9-6 comprises the amino acidsequence of SEQ ID NO:2.
 27. The method of claim 15 wherein said testagent is an organic compound.
 28. The method of claim 15 wherein saidtest agent is an antibody or antigen-binding fragment of an antibody.29. The method of claim 15 wherein said test agent is a peptide.
 30. Themethod of claim 15 wherein said test agent is a nucleic acid.
 31. Themethod of claim 20 wherein said recombinant cell comprises an exogenousnucleic acid encoding said GPR-9-6.
 32. The method of claim 4 whereinsaid recombinant cell comprises an exogenous nucleic acid encoding saidGPR-9-6.
 33. The method of claim 3 wherein the formation of a complexbetween said TECK and said GPR-9-6 is detected or measured by monitoringexocytosis, inflammatory mediator release, respiratory burst or integrinactivation induced upon binding of said TECK to the protein comprisingGPR-9-6 expressed on said cell.
 34. The method of claim 19 wherein theformation of a complex between said TECK and said GPR-9-6 is detected ormeasured by monitoring exocytosis, inflammatory mediator release,respiratory burst or integrin activation induced upon binding of saidTECK to the protein comprising GPR-9-6 expressed on said cell.
 35. Amethod of detecting or identifying an agent which binds a mammalianGPR-9-6 comprising combining: a) TECK, b) a test agent, and c) acomposition comprising a protein, said protein comprising GPR-9-6, underconditions suitable for binding of said TECK to said GPR-9-6, whereinsaid GPR-9-6 is recognized by mAb 3C3 (ATCC Accession No. HB-12653) andbinds TECK; and detecting or measuring the formation of a complexbetween said TECK and said GPR-9-6, wherein a decrease in the formationof said complex relative to a suitable control indicates that said testagent binds a mammalian GPR-9-6.
 36. The method of claim 35 wherein saidTECK is labeled with a label selected from the group consisting of aradioisotope, an epitope, an affinity label, an enzyme, a fluorescentgroup and a chemiluminescent group.
 37. The method of claim 35 whereinsaid composition comprising a protein is a cell that expresses saidprotein comprising GPR-9-6.
 38. The method of claim 37 wherein said cellis a recombinant cell.
 39. The method of claim 37 wherein said cell is acell line.
 40. The method of claim 39 wherein said cell line is selectedfrom the group consisting of MOLT-4 and MOLT-13.
 41. The method of claim37 wherein the formation of a complex between said TECK and said GPR-9-6is detected or measured by monitoring chemotaxis or Ca²⁺ flux inducedupon binding of said TECK to the protein comprising GPR-9-6 expressed onsaid cell.
 42. The method of claim 35 wherein said compositioncomprising a protein is a membrane preparation of a cell that expressessaid protein comprising GPR-9-6.
 43. The method of claim 35 wherein saidGPR-9-6 is a human GPR-9-6.
 44. The method of claim 35 wherein saidGPR-9-6 comprises the amino acid sequence of SEQ ID NO:2.
 45. The methodof claim 35 wherein said test agent is an organic compound.
 46. Themethod of claim 35 wherein said test agent is an antibody orantigen-binding fragment of an antibody.
 47. The method of claim 35wherein said test agent is a peptide.
 48. The method of claim 35 whereinsaid test agent is a nucleic acid.
 49. The method of claim 38 whereinsaid recombinant cell comprises an exogenous nucleic acid encoding saidGPR-9-6.
 50. The method of claim 37 wherein the formation of a complexbetween said TECK and said GPR-9-6 is detected or measured by monitoringexocytosis, inflammatory mediator release, respiratory burst or integrinactivation induced upon binding of said TECK to the protein comprisingGPR-9-6 expressed on said cell.